Transgenic expression of a fungal endo-polygalacturonase increases plant resistance to pathogens and reduces auxin sensitivity

Abstract

Polygalacturonases (PGs), enzymes that hydrolyze the homogalacturonan of the plant cell wall, are virulence factors of several phytopathogenic fungi and bacteria. On the other hand, PGs may activate defense responses by releasing oligogalacturonides (OGs) perceived by the plant cell as host-associated molecular patterns. Tobacco (Nicotiana tabacum) and Arabidopsis (Arabidopsis thaliana) plants expressing a fungal PG (PG plants) have a reduced content of homogalacturonan. Here, we show that PG plants are more resistant to microbial pathogens and have constitutively activated defense responses. Interestingly, either in tobacco PG or wild-type plants treated with OGs, resistance to fungal infection is suppressed by exogenous auxin, whereas sensitivity to auxin of PG plants is reduced in different bioassays. The altered plant defense responses and auxin sensitivity in PG plants may reflect an increased accumulation of OGs and subsequent antagonism of auxin action. Alternatively, it may be a consequence of perturbations of cellular physiology and elevated defense status as a result of altered cell wall architecture.

Figure 1.

Resistance of PG plants to fungal infection. Development of symptoms in tobacco (A and B) and Arabidopsis (C and D) untransformed (WT) and PG plants inoculated with B. cinerea is shown. Fully expanded leaves from untransformed tobacco plants (WT) from three independent transgenic lines expressing PG (PG5, PG7, and PG16) from plants overexpressing bean PvPGIP2 (PGIP2) or generated by crossing line PG16 with the line expressing PvPGIP2 (PG16×PGIP2) were inoculated with B. cinerea. Lesion area (A) and percentage of expanding lesions (B) were measured after 6 d. Bars in A indicate average lesion area ± se (se; n > 36); this experiment was repeated four times with similar results. Bars in B represent the average percentage of spreading lesions ± se of five experiments (n > 36 in each experiments). Adult rosette leaves from untransformed Arabidopsis plants (WT) and from transgenic plants expressing PG (PG1 and PG5) or an inactive version of the A. niger PGII (PG201) were inoculated with B. cinerea, and lesion size was determined after 2 d (C), whereas the number of expanding lesions was determined after 3 d (D). Bars in C indicate the average lesion area ± se (n > 12); this experiment was repeated three times with similar results. Bars in D represent the average percentage of spreading lesions ± se of three experiments (n > 12 in each experiment). Different letters represent data sets significantly different, according to ANOVA analysis followed by Tukey's test (P < 0.01).

Figure 2.

Resistance of tobacco PG plants to bacterial infection. Disease symptoms of (from top to bottom and from left to right) tobacco untransformed plants (WT) and transgenic PG5, PG7, PG16, PG16×PGIP2, and PGIP2 plants injected with a virulent strain of P. syringae pv tabaci. Inoculated leaves of the indicated genotypes were photographed 5 d after inoculation. This experiment was repeated twice with similar results.

Figure 3.

Accumulation of H₂O₂ in PG plants. A, DAB staining of detached leaves from (from left to right) tobacco untransformed (WT) and transgenic PG5, PG7, PG16, PG×PGIP2, and PGIP2 plants. B, DAB staining of detached leaves from (from left to right) Arabidopsis untransformed (WT) and transgenic PG201, PG5, and PG1 plants.

Figure 4.

Peroxidase activity in PG plants. A, Peroxidase activity in total protein extracts from leaves of tobacco untransformed (WT) and transgenic PG5, PG7, PG16, PGIP2, and PG×PGIP2 plants. B, Peroxidase activity in IWFs (black bars) and intracellular proteins extracted after recovery of IWFs (white bars) from the same tobacco lines shown in A. C, Peroxidase activity in total protein extracts from leaves of Arabidopsis untransformed (WT) and transgenic PG201, PG5, and PG1 plants. Bars indicate the average activity, expressed as enzymatic units per milligram of total proteins, of three samples ± se. Different letters represent data sets significantly different, according to ANOVA analysis followed by Tukey's test (P < 0.01).

Figure 5.

Glucanase activity in PG plants. A, Levels of β-1,3-glucanase activity, expressed as enzymatic units per milligram of total proteins, in leaves of tobacco untransformed (WT) and transgenic PG5, PG7, PG16, PG×PGIP2, and PGIP2 plants. Bars indicate average activity of three independent samples ± se. B, Levels of β-1,3-glucanase activity, expressed as enzymatic units per milligram of total proteins, in leaves of Arabidopsis untransformed (WT) and transgenic PG201, PG5, and PG1 plants. Bars indicate the average activity of three independent samples ± se. Different letters represent data sets significantly different, according to ANOVA analysis followed by Tukey's test (P < 0.01).

Figure 6.

Expression of defense genes in PG plants after infection with B. cinerea. A, Fully expanded leaves from tobacco untransformed (WT) and transgenic PG16 (PG) plants were inoculated with B. cinerea and total RNA was extracted at the indicated time points (dpi). The RNA gel blot was hybridized with the indicated probes. Equal loading was verified by methylene blue staining of ribosomal RNA. B to D, Fully expanded leaves from Arabidopsis untransformed (WT, white bars) or transgenic PG1 (PG, black bars) plants were inoculated with B. cinerea, and total RNA was extracted at the indicated time points (dpi). The expression of AtPGIP1 (B), PR-1 (C), and PDF1.2 (D) was analyzed by real-time RT-PCR and normalized using the expression of UBQ5 in each sample. The insets in C and D show gene expression in untreated leaves (n.d., not detectable). Bars represent the average expression ± sd of two replicates.

Figure 7.

Regulation of defense gene expression by OGs in tobacco plants. A and B, Real-time RT-PCR analysis of the expression of EAS1/2 (A) and POX (B) in untransformed tobacco leaf explants treated for the indicated times with water (control, white bars) or OGs (OG, black bars). The expression of each gene was normalized using the expression of the actin gene Tob66 in each sample. Bars represent the average gene expression ± sd of two replicates.

Figure 8.

Regulation of defense gene expression by OGs in Arabidopsis plants. A to C, Real-time RT-PCR analysis of the expression of AtPGIP1 (A), PR-1 (B), and PDF1.2 (C) in untransformed Arabidopsis seedlings treated for the indicated times with OGs. The expression of each gene was normalized using the expression of UBQ5 in each sample. Bars represent the average gene expression ± sd of two replicates.

Figure 9.

Susceptibility to B. cinerea in tobacco PG plants treated with auxin. A, Leaf discs from tobacco untransformed (WT, white bars) or transgenic PG16 (PG, black bars) plants were incubated for 3 h in liquid medium containing water (control) or 100 μm IAA (IAA) and inoculated with B. cinerea. Lesion area was measured after 24 h. Bars indicate average area ± se of at least 10 lesions. Asterisks indicate statistically significant difference between lesions in wild-type and transgenic plants (***, P < 0.01). This experiment was repeated three times with similar results. B, Leaf discs from untransformed tobacco plants were incubated for 3 h in liquid medium containing water (control), 100 μm IAA (IAA), 200 μg mL⁻¹ OGs (OG), or both (OG + IAA) and inoculated with B. cinerea. Lesion area was measured after 24 h. Bars indicate the average area ± se of at least 10 lesions. Different letters indicate data sets significantly different, according to ANOVA followed by Tukey's test (P < 0.05). This experiment was repeated twice with similar results.

Figure 10.

Auxin sensitivity of tobacco PG plants. A, Leaf explants from tobacco untransformed (WT, white squares) and transgenic PG16 (PG, black squares) plants were treated with the indicated concentrations of IAA for 15 d and the number of explants forming roots was measured. Each data point represents the average percentage of explants forming roots ± sd calculated in three independent experiments (n > 8 in each experiment). B, Length of primary roots of tobacco untransformed (WT) and transgenic PG16 (PG) plants grown for 12 d on solid medium containing the indicated IAA concentrations. Bars represent the average length of at least eight plants ± se. Asterisks indicate statistically significant difference between treated and control samples, according to the Student's t test (P < 0.01). This experiment was repeated twice with similar results.

Figure 11.

Ethylene production in tobacco PG leaf explants. Leaf explants from tobacco untransformed (WT, white bars) and transgenic PG16 (PG, black bars) plants were incubated with water, 250 μm ACC, or 100 μm IAA (IAA) in sealed flasks. Ethylene accumulating in the flask was determined by gas chromatography. Bars indicate ethylene concentration after 24 h of treatment. No detectable accumulation of ethylene could be observed in control samples. This experiment was repeated twice with similar results.