A R2R3-MYB gene, AtMYB30, acts as a positive regulator of the hypersensitive cell death program in plants in response to pathogen attack

Abstract

Hypersensitive response (HR) is a programmed cell death that is commonly associated with disease resistance in plants. Among the different HR-related early induced genes, the AtMYB30 gene is specifically, rapidly, and transiently expressed during incompatible interactions between Arabidopsis and bacterial pathogens. Its expression was also shown to be deregulated in Arabidopsis mutants affected in the control of cell death initiation. Here, we demonstrate that overexpression in Arabidopsis and tobacco of AtMYB30 (i) accelerates and intensifies the appearance of the HR in response to different avirulent bacterial pathogens, (ii) causes HR-like responses to virulent strains, and (iii) increases resistance against different bacterial pathogens, and a virulent biotrophic fungal pathogen, Cercospora nicotianae. In antisense AtMYB30 Arabidopsis lines, HR cell death is strongly decreased or suppressed in response to avirulent bacterial strains, resistance against different bacterial pathogens decreased, and the expression of HR- and defense-related genes was altered. Taken together, these results strongly suggest that AtMYB30 is a positive regulator of hypersensitive cell death.

Figure 1

Phenotypes of AtMYB30 sense and antisense Arabidopsis and tobacco lines in response to inoculation with avirulent or virulent bacteria. Symptoms observed in Arabidopsis sense, wild-type, and antisense lines: (A) 24 h postinoculation with DC3000/avrRpm1 (10⁷ cfu/ml), (B) 7 days postinoculation with DC3000 (10⁷ cfu/ml), (C) 24 h postinoculation with Xcc147 (10⁸ cfu/ml), and (D) 48 h postinoculation with Xcc8004 (10⁸ cfu/ml). (E and F) Phenotypes of tobacco transgenic lines in response to inoculation with R. solanacearum. Different inocula were used for the avirulent strain (GMI1000), ranging from 5.10⁷ cfu/ml (no. 1), 10⁷ cfu/ml (no. 2), to 5.10⁶ cfu/ml (no. 3); the virulent strain (K60) was inoculated at 5.10⁷ cfu/ml (no. 4), and a control inoculation was performed with water (no. 5).

Figure 2

Effect of overexpression and antisense depletion of AtMYB30 on cell death occurring in response to bacterial avirulent pathogen attack. (A and B) Evaluation of cell death in transgenic Arabidopsis leaves after inoculation with the avirulent strain DC3000/avrRpm1 of P. syringae at 10⁷ cfu/ml. Uptake of Evans blue by leaves from wild-type plants (○), sense transgenic plants (A) (pBIM131–20A, ●; pBIM131–17A, ▴) and antisense transgenic plants (B) (pBIW13–1A, ●; pBIW13–1B, ▴). Data are expressed as OD units, and means and SD from three replicates in two independent experiments are given. (C and D) Reverse transcription–PCR analysis of AtMYB30 and β-tubulin mRNA accumulation in plant extracts from wild-type (WS) plants, control plants (23A), sense lines (11B, 19A, 15A, 18B, 20A, and 17A), and antisense lines (18B, 10B, 9A, 1B, 1A, and 4B).

Figure 3

Effect of overexpression of AtMYB30 on hydroxy fatty acid production in response to bacterial pathogen attack. HPLC analysis of hydroxy fatty acids extracted from wild-type and sense transgenic plants (pBIM131–40A). (A and B) HPLC traces of wild-type leaf extracts (Lower) and sense transgenic leaf extracts (Upper), 22 h after inoculation with an avirulent strain (5.10⁷ cfu/ml) (A) or 76 h after inoculation with a virulent strain (5.10⁷ cfu/ml) (B) of R. solanacearum. The various hydroxy fatty acids isomers of 18:2 and 18:3 fatty acids were identified as described (21) and quantified with reference to the internal standard 15-hydroxy-11,13(Z,E) eicosadienoic acid. (C–E). Changes in 9-HOTE (filled column), X (empty column), and Y (dotted column) compound levels of sense and wild-type tobacco leaves 22 h (C) and 72 h (D) after inoculation with an avirulent strain, or 76 h after inoculation with a virulent strain (E) of R. solanacearum. Mean and SD from three replicates of the same experiment are given.

Figure 4

Effects of AtMYB30 overexpression on hsr and defense-related gene expression. (A) Time course of hsr203J promoter activation in transgenic tobacco plants resulting (or not, control) from a cross (F₂ plants whose genotype has been determined) between an AtMYB30 sense line (pBIM131–40A) and a line containing the hsr203J promoter-GUS fusion (13), after inoculation with different isolates of R. solanacearum (5.10⁷ cfu/ml) or water. GUS activity was measured in extracts of two leaves (equivalent of four replicates) after inoculation with the avirulent strain (○), virulent strain (□), and water (▵) from the control line (open symbols) or the F₂ line (filled symbols). (B and C) Alterations in the expression of hsr genes and PR genes in transgenic sense and control tobacco plants (B) and in transgenic sense, antisense, and control Arabidopsis plants (C). Total RNA was isolated at different times after inoculation with Ralstonia (5.10⁷ cfu/ml) (B) or P. syringae DC3000/avrRpm1 (10⁷ cfu/ml) (C) from the leaves.

Figure 5

Disease resistance of transgenic Arabidopsis and tobacco expressing constitutively AtMYB30 to different virulent and avirulent pathogens. (A and B) Measurement of in planta growth of P. syringae pv. tomato in wild-type Arabidopsis plants (○), sense transgenic plants (pBIM131–20A, ■), or antisense plants (pBIW13–1A, ▴), inoculated (A) with the avirulent strain DC3000/avrRpm1 (10⁷ cfu/ml) or (B) with the virulent strain DC3000 (10⁷ cfu/ml). Data points represent the mean of three replicate experiments and SDs are shown. (C) Growth of C. nicotianae in wild-type tobacco plants, control plants (pBI111–23A), or sense transgenic plants (pBIM131–40A). Twelve to 20 independent measurements of fungal growth were made on 10 leaves from five plants at each time point, and the maximal and minimal data are indicated in cm². Statistically significant differences have been measured between the sense line and the control line [t test, 0.01 < P < 0.05 (14 days postinoculation, dpi); P < 0.0001 (16 dpi), P < 0.0001 (21 dpi)] and between the sense line and the wild-type line [t test, P < 0.0001 (14 dpi); P < 0.0001 (16 dpi); P < 0.0001 (21 dpi)]. (D) Disease symptoms caused by C. nicotianae 14 days after inoculation in the wild-type, control, and sense lines. The black circle indicates the inoculated zone.

Figure 6

Proposed model for the role of AtMYB30 in cell death initiation. AtMYB30 gene is specifically, rapidly, and transiently expressed in response to avirulent pathogens. Overexpression of AtMYB30 leads to an acceleration and enhancement of HR cell death, whereas in antisense AtMYB30 lines, HR cell death is strongly decreased or suppressed. AtMYB30 overexpression does not confer a lesion mimic phenotype, so the expression of AtMYB30 is not sufficient by itself to induce the hypersensitive cell death program. AtMYB30 probably acts in cooperation with other factor(s) (F) for initiation of the program.