Spermine is a salicylate-independent endogenous inducer for both tobacco acidic pathogenesis-related proteins and resistance against tobacco mosaic virus infection

Abstract

Intercellular spaces are often the first sites invaded by pathogens. In the spaces of tobacco mosaic virus (TMV)-infected and necrotic lesion-forming tobacco (Nicotiana tabacum L.) leaves, we found that an inducer for acidic pathogenesis-related (PR) proteins was accumulated. The induction activity was recovered in gel-filtrated fractions of low molecular mass with a basic nature, into which authentic spermine (Spm) was eluted. We quantified polyamines in the intercellular spaces of the necrotic lesion-forming leaves and found 20-fold higher levels of free Spm than in healthy leaves. Among several polyamines tested, exogenously supplied Spm induced acidic PR-1 gene expression. Immunoblot analysis showed that Spm treatment increased not only acidic PR-1 but also acidic PR-2, PR-3, and PR-5 protein accumulation. Treatment of healthy tobacco leaves with salicylic acid (SA) caused no significant increase in the level of endogenous Spm, and Spm did not increase the level of endogenous SA, suggesting that induction of acidic PR proteins by Spm is independent of SA. The size of TMV-induced local lesions was reduced by Spm treatment. These results indicate that Spm accumulates outside of cells after lesion formation and induces both acidic PR proteins and resistance against TMV via a SA-independent signaling pathway.

Figure 1

Inducer activity for PR gene expression in gel-filtrated fractions of the intercellular fluid from TMV-infected tobacco leaves. Intercellular fluid of TMV-infected and necrotic lesion-forming tobacco leaves was concentrated and applied to a Sephadex G-15 column. The gel-filtrated fractions were tested for their ability to induce acidic PR-1a gene expression in leaf discs of PR1a-GUS transgenic tobacco plants. GUS activity in the leaf discs was determined 3 d after treatment with each fraction. The underlining bar shows the fractions to which authentic Spm was eluted. The experiment was repeated twice with similar results. FW, Fresh weight.

Figure 2

Induced accumulation of Spm in the intercellular spaces of local lesion-forming tobacco leaves. Free Spm in the intercellular fluid was quantified after TMV or mock inoculation. Data are the mean values of triplicate samples. Error bars indicate sd. The experiment was repeated twice with similar results. FW, Fresh weight.

Figure 3

Induction of PR1a-GUS gene expression by PAs. GUS activity in the leaf discs of PR1a-GUS transgenic tobacco plants was determined 3 d after the treatment with various PAs. For a positive control, leaf discs were treated with 50 μm SA (pH 7.0). Data are the mean values of triplicate samples. Error bars indicate sd. The experiment was repeated twice with similar results. FW, Fresh weight.

Figure 4

Induction of GUS activity by Spm at various pH levels. Leaf discs of PR1a-GUS plants were treated with free Spm in water (pH 8.5, 8.9, and 9.4 at 50, 150, and 500 μm, respectively) (A), with Spm neutralized with HCl (pH 7.0) (B), or with Spm in the presence of 10 mm Mes buffer (pH 5.5) (C). GUS activity in the leaf discs was determined 3 d after the treatment. For a positive control, leaf discs treated with 50 μm SA (pH 7.0) were used. Data are the mean values of triplicate samples. Error bars indicate sd. The experiment was repeated twice with similar results. FW, Fresh weight.

Figure 5

Time-course analysis of PR1a-GUS gene expression induced by Spm and SA. Leaf discs of PR1a-GUS plants were treated with distilled water (DW, –□–), 300 μm Spm (pH 9.2) (–•–), or 50 μm SA (–○–), and GUS activity in the leaf discs was determined after 0 to 3 d. Data are the mean values of triplicate samples. Error bars indicate sd. The experiment was repeated twice with similar results. FW, Fresh weight.

Figure 6

Immunoblot analysis of acidic PR-1, PR-2, PR-3, and PR-5 proteins. Leaf pieces were treated with 150, 300, or 500 μm Spm for 3 d. After tissue homogenization, soluble protein was extracted. To evaluate acidic PR protein induction, 50 μm SA-treated leaves and leaves 3 d after TMV infection were also extracted as positive controls. Fifteen micrograms of protein (equivalent to 3 mg of fresh leaf material) was subjected to SDS-PAGE (A) or native-PAGE (B). Immunodetection was performed with specific antibodies against individual purified PR proteins (see Methods). Each PR protein was identified by comparison of its mobility with that of M_r markers or standard samples of purified PR proteins. BCs, Basic chitinases. The experiment was repeated twice with similar results.

Figure 7

Effect of SA treatment on the amount of Spm recovered from the intercellular fluid. Free Spm was quantified in the intercellular fluid of tobacco leaves that were incubated for 2 or 4 d in 500 μm SA solution or in distilled water (DW). Data are the mean values of triplicate samples. Error bars indicate sd. The experiment was repeated twice with similar results. FW, Fresh weight.

Figure 8

Effect of exogenously supplied Spm on the accumulation of SA and SAG. SA (A) and SAG (B) were quantified 2 or 4 d after incubation with 300 μm Spm (▪) or distilled water (□). As a positive control, leaves were used 4 d after TMV inoculation (▧). Data are the mean values of triplicate samples. Error bars indicate sd. The experiment was repeated twice with similar results. FW, Fresh weight.

Figure 9

Induced resistance against TMV infection by Spm. Detached tobacco leaves were inoculated with TMV 2 d after the treatment with 300 μm Spm solution or distilled water and incubated for a further 4 d at 22°C. Then, the lesion size was measured in three areas: apical (□), middle (▨), and basal (▪) sections were evaluated using enlarged photocopies. At least 100 lesions from four leaves were measured for each section. Data are mean values. Error bars indicate sd. Evaluation by a Student's t test resulted in P < 0.001 for each section between Spm- and water-treated leaves. Lesions that developed in basal parts of preincubated leaves are shown in B. The experiment was repeated twice with similar results.

Figure 10

Induction of resistance against TMV by Spm. Detached tobacco leaves were treated with various concentrations of Spm for 2 d and inoculated with TMV. The diameter of developed local lesions was measured after 4 additional d of incubation. The measurement was performed separately in three areas: apical (□), middle (▨), and basal (▪). At least 100 lesions from 4 leaves were measured for each section. Data are mean values. Error bars indicate sd.

Figure 11

Proposed model for the pathway signaling acidic PR protein induction and acquisition of resistance. Spm accumulated in the intercellular spaces of TMV-infected tobacco leaves induced a high level of acidic PR protein expression and conferred resistance to further infection by TMV. This induction was not affected by SA. See text for details.