Abscisic acid determines basal susceptibility of tomato to Botrytis cinerea and suppresses salicylic acid-dependent signaling mechanisms

Abstract

Abscisic acid (ABA) is one of the plant hormones involved in the interaction between plants and pathogens. In this work, we show that tomato (Lycopersicon esculentum Mill. cv Moneymaker) mutants with reduced ABA levels (sitiens plants) are much more resistant to the necrotrophic fungus Botrytis cinerea than wild-type (WT) plants. Exogenous application of ABA restored susceptibility to B. cinerea in sitiens plants and increased susceptibility in WT plants. These results indicate that ABA plays a major role in the susceptibility of tomato to B. cinerea. ABA appeared to interact with a functional plant defense response against B. cinerea. Experiments with transgenic NahG tomato plants and benzo(1,2,3)thiadiazole-7-carbothioic acid demonstrated the importance of salicylic acid in the tomato-B. cinerea interaction. In addition, upon infection with B. cinerea, sitiens plants showed a clear increase in phenylalanine ammonia lyase activity, which was not observed in infected WT plants, indicating that the ABA levels in healthy WT tomato plants partly repress phenylalanine ammonia lyase activity. In addition, sitiens plants became more sensitive to benzo(1,2,3)thiadiazole-7-carbothioic acid root treatment. The threshold values for PR1a gene expression declined with a factor 10 to 100 in sitiens compared with WT plants. Thus, ABA appears to negatively modulate the salicylic acid-dependent defense pathway in tomato, which may be one of the mechanisms by which ABA levels determine susceptibility to B. cinerea.

Figure 1

Effect of Glc and phosphate concentrations on the infection of B. cinerea on tomato. Detached tertiary leaves (A) or tertiary leaves of intact plants (B) were infected by a droplet solution. Ten droplets each containing 4 μL of spore suspension were placed on a tomato leaf surface. The infection was evaluated at several time points after infection by counting the number of B. cinerea lesions spreading out of the initial inoculation droplets on each leaf. The inoculation solutions tested in this work were: ●, 0.1 m Glc, 67 mm KH₂PO₄ (pH 5), and 10⁶ spores mL⁻¹; ▪, 0.05 m Glc, 33 mm KH₂PO₄ (pH 5), and 10⁶ spores mL⁻¹; and ▴, 0.01 m Glc, 6.7 mm KH₂PO₄ (pH 5), and 10⁶ spores mL⁻¹. White signs show the infection development without adding phosphate to the inoculation solutions. Data are means of three experiments containing 12 leaves per treatment.

Figure 2

A, Influence of endogenous ABA concentrations on spreading of B. cinerea in tomato 4 d after infection. Tertiary WT (tomato cv Moneymaker) and sitiens leaves (tomato cv Moneymaker) were detached from 5-week-old tomato plants and infected with 10 droplets of a 4-μL spore suspension containing 10⁶ spores mL⁻¹, 0.01 m Glc, and 6.7 mm KH₂PO₄. Infection was evaluated 4 d after infection by counting the number of spreading lesions on each leaf. Data are means of three experiments containing 12 leaves per treatment. B shows an infected WT leaf (tomato cv Moneymaker) with four spreading lesions of six lesions. C shows a resistant sitiens leaf (tomato cv Moneymaker) with no spreading lesions. Bars with different letters are significantly different with P = 0.05 after a logistic regression.

Figure 3

Effect of exogenous ABA fed to petioles of 5-week-old of sitiens (A) and WT (B) leaves (tomato cv Moneymaker) on an infection with B. cinerea. Tertiary leaves were detached and placed for 16 h in ABA solutions varying from 1 to 100 μm. ABA solutions were prepared from 1 mL of 10 mm stock solution of ABA in ethanol. Ethanol concentrations varied from 0.1% to 0.001% (v/v), respectively, in the final solutions. Control leaves were fed with water containing 0.1% (v/v) ethanol. Inoculation solutions contained 0.01 m Glc, 6.7 mm KH₂PO₄, and 10⁶ spores mL⁻¹. Infection was scored 4 d after inoculation by counting the number of spreading B. cinerea lesions on each leaf. Data are means of three experiments containing 12 leaves per treatment. Bars with different letters are significantly different with P = 0.05 after a logistic regression.

Figure 4

Influence of endogenous ABA concentrations on spreading of B. cinerea in intact tomato plants. Tertiary leaves of WT (tomato cv Moneymaker) and sitiens (tomato cv Moneymaker) plants were infected with 10 droplets of 4 μL of spore suspension containing 10⁶ spores mL⁻¹, 0.01 m Glc, and 6.7 mm KH₂PO₄. Data are means of two experiments containing five plants (10 leaves) per treatment. Infections were evaluated 4 d after inoculation by counting the number of spreading B. cinerea lesions on each leaf. Bars with different letters are significantly different with P = 0.05 after a logistic regression.

Figure 5

A, Effect of endogenous JA concentrations on the infection of B. cinerea in tomato. Tertiary leaves of WT (tomato cv Castlemart) and def1 tomato mutants (tomato cv Castlemart) were infected with 10 droplets of 4 μL of spore suspension containing 10⁶ spores mL⁻¹, 0.01 m Glc, and 6.7 mm KH₂PO₄. Data are means of two experiments containing 10 leaves per treatment. Infections were evaluated 4 d after inoculation by counting the number of spreading B. cinerea lesions on each leaf. Bars with different letters are significantly different with P = 0.05 after a logistic regression. B, Effect of exogenous ± JA feeding through petioles of tomato leaves from 5-week-old plants. Leaves were infected with 10 droplets of 4 μL of spore suspension containing 10⁶ spores mL⁻¹, 0.01 m Glc, and 6.7 mm KH₂PO₄. Data are means of two experiments containing 10 leaves per treatment. Bars with different letters are significantly different with P = 0.05 after a logistic regression.

Figure 6

A, Influence of endogenous SA on spreading of B. cinerea in the detached leaf assay. Tertiary WT (tomato cv Moneymaker) and NahG leaves (tomato cv Moneymaker) were infected with 10 droplets of 4 μL of spore suspension containing 10⁶ spores mL⁻¹, 0.01 m Glc, and 6.7 mm KH₂PO₄. Data are means of two experiments each composed of 10 leaves per treatment. Infection was evaluated 4 d after inoculation by counting the number of spreading B. cinerea lesions on each leaf. Bars with different letters are significantly different with P = 0.05 after a logistic regression. B, Relative induction of PAL activity in 5-week-old WT (tomato cv Moneymaker) and sitiens leaves (tomato cv Moneymaker) 16 h after infection with B. cinerea with an infection solution of 10⁶ spores mL⁻¹, 0.01 m Glc, and 6.7 mm KH₂PO₄. Values for PAL activity were obtained by dividing values for PAL activity 16 h after infection by values for PAL activity of control leaves. Bars represent the average of 10 individual tomato leaves of sitiens and WT plants. Data represent two independent experiments. Data were analyzed by an ANOVA analysis. Bars with different letters are significantly different with P = 0.05.

Figure 7

A. Effect of BTH (BION) root application on spreading of B. cinerea lesions on the third pair of 5-week-old WT tomato leaves (tomato cv Moneymaker). Leaves were infected with 10 droplets of 4 μL of spore suspension containing 10⁶ spores mL⁻¹, 0.01 m Glc, and 6.7 mm KH₂PO₄ 25 d after the last BTH treatment. Data are means of three experiments with each 12 leaves per treatment. Infections were evaluated 4 d after inoculation by counting the number of spreading B. cinerea lesions on each leaf. Bars with different letters are significantly different with P = 0.05 after a logistic regression. B, Induction of PR1a gene expression in 5-week-old leaves of WT tomato plants after BTH root application. Each lane contains 30 μg of total RNA and PR1a was detected via digoxygenin (DIG)-labeled cDNA probes. BTH concentrations are represented in milligrams per kilogram. Detection limit of PR1a was determined to be 0.5 pg. An 18S rRNA probe was used as a constitutive probe to verify for equal RNA loading and transfer.

Figure 8

Influence of inoculum aggressiveness on the resistance of sitiens leaves (tomato cv Moneymaker) to B. cinerea. Tertiary leaves of WT and sitiens plants were infected with 10 droplets of 4 μL of spore suspension containing different Glc and phosphate concentrations. Data are means of three experiments with each 12 leaves per treatment. Infections were evaluated 4 d after infection by counting the number of spreading B. cinerea lesions on each leaf. Bars with different letters are significantly different with P = 0.05 after a logistic regression.

Figure 9

A, Effect of BTH root application on spreading of B. cinerea lesions on the third pair of sitiens tomato leaves (tomato cv Moneymaker). Leaves were infected with 10 droplets of 4 μL of spore suspension containing 10⁶ spores mL⁻¹, 0.05 m Glc, and 33 mm KH₂PO₄ 25 d after the last BTH treatment. Data are means of three experiments with each 12 leaves per treatment. Infections were evaluated 4 d after infection by counting the number of spreading B. cinerea lesions on each leaf. Bars with different letters are significantly different with P = 0.05 after a logistic regression. B, Induction of PR1a gene expression in 5-week-old leaves of sitiens tomato plants after BTH root application. Each lane contains 30 μg of total RNA and PR1a was detected via DIG-labeled cDNA probes. An 18S rRNA probe was used as a constitutive probe to verify for equal RNA loading and transfer.