Defense activation and enhanced pathogen tolerance induced by H2O2 in transgenic tobacco

Abstract

Transgenic tobacco deficient in the H2O2-removing enzyme catalase (Cat1AS) was used as an inducible and noninvasive system to study the role of H2O2 as an activator of pathogenesis-related (PR) proteins in plants. Excess H2O2 in Cat1AS plants was generated by simply increasing light intensities. Sustained exposure of Cat1AS plants to excess H2O2 provoked tissue damage, stimulated salicylic acid and ethylene production, and induced the expression of acidic and basic PR proteins with a timing and magnitude similar to the hypersensitive response against pathogens. Salicylic acid production was biphasic, and the first peak of salicylic acid as well as the peak of ethylene occurred within the first hours of high light, which is long before the development of tissue necrosis. Under these conditions, accumulation of acidic PR proteins was also seen in upper leaves that were not exposed to high light, indicating systemic induction of expression. Short exposure of Cat1AS plants to excess H2O2 did not cause damage, induced local expression of acidic and basic PR proteins, and enhanced pathogen tolerance. However, the timing and magnitude of PR protein induction was in this case more similar to that in upper uninfected leaves than to that in hypersensitive-response leaves of pathogen-infected plants. Together, these data demonstrate that sublethal levels of H2O2 activate expression of acidic and basic PR proteins and lead to enhanced pathogen tolerance. However, rapid and strong activation of PR protein expression, as seen during the hypersensitive response, occurs only when excess H2O2 is accompanied by leaf necrosis.

Figure 1

Expression of acidic PR proteins in Cat-deficient tobacco and SA requirement for induction. Cat1AS and control plants precultivated at LL were exposed to HL for 2 days. (A) Western blot analysis showing high expression of acidic PR proteins in Cat1AS plants exposed to HL. Cat1AS × Xnc is a cross of homozygous Cat1AS and Xanthi nc lines, whereas Cat1AS × NahG is a cross of homozygous Cat1AS and a transgenic Xanthi nc line containing the NahG gene of Pseudomonas putida, which codes for an SA hydroxylase (9). (B) Time course analysis of SA and SA β-glucoside (SAG) accumulation in Cat1AS plants after HL induction. FW, fresh weight.

Figure 2

Systemic expression of PR-1 in local and distal leaves of Cat1AS plants after HL induction. Cat1AS and control plants precultivated at LL were exposed to HL for 2 days and then returned for 2 weeks at LL before harvest. (Upper Left) Half of a leaf was covered with foil during the HL treatment for analysis of LAR gene expression. Parts of the leaf that had been covered during the HL exposure showed no visible necrosis, whereas the exposed parts of the same leaf developed necrotic lesions within 2 days after the HL treatment. The immunoblot shows PR-1 accumulation in HL-exposed and covered parts of the same leaf. The presence of visible leaf damage at the time of harvest is indicated. (Lower Left) A top leaf was entirely covered to shield it from the light during the HL treatment to assess SAR gene expression. Samples were taken from a lower HL-exposed leaf that showed severe necrosis and from the HL-protected upper leaf, which was undamaged. Western blot analysis is as in the panel above. The presence of leaf damage at the time of harvest is indicated. (Right) Photographs showing representative leaves of Cat1AS plants harvested after HL treatment for analysis of LAR (Top) and SAR (damaged lower leaf and intact upper leaf in Middle and Bottom, respectively) gene expression.

Figure 3

Western analysis showing the effect of HL on the expression of GPx and bPR-2 in Cat1AS plants. Cat1AS and control plants precultivated at LL were exposed to HL for 2 days. Samples were harvested 2 days after HL initiation for analysis of protein expression in HL-exposed tissue (Left), or after 2 additional weeks at LL for analysis of LAR (Center) and SAR gene expression (Right). The presence of visible leaf damage at the time of harvest is indicated. The experimental design for analysis of LAR and SAR gene expression was as described in Fig. 2.

Figure 4

Time course analysis of ethylene formation in Cat1AS plants after exposure to HL. Cat1AS and control plants precultivated at LL were exposed to HL for 2 days and leaf samples were taken for ethylene quantitation at the times indicated.

Figure 5

Necrosis-independent expression of defense proteins in Cat1AS plants. (A) Western blot analysis with PR-1, bPR-2, and GPx antibodies. Cat1AS and control plants were exposed to HL for various times (2–8 h) and then returned to LL for 2 weeks before leaf sampling. Expression of PR-1, bPR-2, and GPx was induced by 4 h of HL in Cat1AS, whereas necrosis required 8 h of exposure. PR-1 expression in the control line was not increased by HL. bPR-2 and GPx showed HL induction in the control line but not to the same level as in Cat1AS. Leaf damage was assessed at the time of harvest: no damage indicates that none of the leaves of that plant had any visible sign of injury. (B) Optical sections of a YOYO-1 iodide-stained leaf from a Cat1AS plant treated with HL for 4 h (Left) or 8 h (Right). YOYO-1 iodide is a nuclear dye that is membrane-impermeant and therefore stains only nuclei of damaged cells. (×700.)

Figure 6

HL-induced protection of Cat1AS plants against Pseudomonas syringae pv. syringae. Cat1AS and control plants were either maintained at LL or exposed to HL for different times as described in the legend of Fig. 5 and returned to LL for 2 weeks. Leaf damage was assessed as described in Fig. 5. Then, Pseudomonas syringae pv. syringae at 10⁶ per ml were inoculated by infiltration in the leaf and bacterial growth was determined after 24 h by plating serial dilutions of homogenate from inoculated tissue on solid medium (22). cfu, colony-forming units. The figure displays a representative result of three repetitions.