Ferulic Acid, But Not All Hydroxycinnamic Acids, Is a Novel T3SS Inducer of Ralstonia solanacearum and Promotes Its Infection Process in Host Plants under Hydroponic Condition

Abstract

Hydroxycinnamic acids (HCAs) are typical monocyclic phenylpropanoids, including cinnamic acid (Cin), coumaric acid (Cou), caffeic acid (Caf), ferulic acid (FA) and their isomers, and involved in the interactions between pathogens and host plants. Here, we focused on the impact of HCAs on expression of type III secretion system (T3SS) in Ralstonia solanacearum. FA significantly induced the expression of the T3SS and some type III effectors (T3Es) genes in hrp-inducing medium, while did not the other HCAs. However, exogenously supplemented FA did not affect the T3SS expression in planta and the elicitation of the hypersensitive response (HR) in tobacco leaves. Consistent with its central roles in pathogenicity, the FA-induced expression of the T3SS led to significant promotion on infection process of R. solanacearum in tomato plants under hydroponics cultivation. Moreover, the FA-induced expression of the T3SS was specifically mediated by the well-characterized signaling cascade PrhA-prhI/R-PrhJ-HrpG-HrpB, independent of the other known regulatory pathways. In summary, our results demonstrated that FA, a novel inducer of the T3SS in R. solanacearum, was able to promote its infection process in host plants under hydroponics condition.

Keywords: Hrp regulon; Ralstonia solanacearum; hydroxycinnamic acids; pathogenesis; type III secretion system.

FIGURE 1

Effect of HCAs on growth of Ralstonia solanacearum in medium. (A) Structure of HCAs; (B) rich medium; (C) hrp-inducing medium. Cells were inoculated into fresh HCAs medium with the ratio as 1% and grown for about 16 h for OD₆₀₀ measurement. HCAs were caffeic acid (Caf), cinnamic acid (Cin), p-coumaric acid (Cou), and ferulic acid (FA). Each test was repeated for at least three independent trials and each trial was repeated in twice. Mean values were averaged and presented with SD (error bars). Statistical significance between HCAs treatment and the control was assessed using a post hoc Dunnett test following ANOVA. Significance level, ^∗ p < 0.05 and ^∗∗p < 0.01.

FIGURE 2

Effect of HCAs on the popA expression in vitro. (A) hrp-inducing medium supplemented with 0.5 mM of each HCAs; (B) hrp-inducing medium supplemented with gradient concentration of FA. Cells were grown in hrp-inducing medium to an OD₆₀₀ of approximately 0.1 and subjected for β-galactosidase assay. The mean values of at least three independent trials are presented in Miller units with SD (error bars). Statistical significance between HCAs (or FA) treatment and the control was assessed using a post hoc Dunnett test following ANOVA. Significance level, ^∗p < 0.05 and ^∗∗p < 0.01.

FIGURE 3

Relative expression of T3Es genes with FA treatment by qRT-PCR. The OE1-1 was grown in hrp-inducing medium (with 0.5 mM of FA or DMSO control) to OD₆₀₀ as about 0.1 and total RNA was isolated for qRT-PCR analysis. Eight representative T3Es genes for selected for determination and the serC gene was used as reference gene for normalization of gene expression. Normalized value with FA treatment was divided with that of control and relative values were presented. Each test was repeated from RNA isolation in at least three independent trials and each trials included four replications. Mean values were averaged and presented with SD (error bars). Statistical significance between FA treatment and the DMSO control was assessed using a post hoc Dunnett test following ANOVA. Significance level, ^∗p < 0.05 and ^∗∗p < 0.01.

FIGURE 4

Relative expression of genes involved in hrp regulation with FA treatment. Cells were grown in hrp-inducing medium to an OD₆₀₀ of approximately 0.1 and subjected for β-galactosidase assay. Normalized value with FA treatment was divided with that of control and relative values were presented. Each test was repeated in at least three independent trails and each trails included two replications. Mean values were averaged and presented with SD (error bars). Statistical significance between FA treatment and the DMSO control was assessed using a post hoc Dunnett test following ANOVA. Significance level, ^∗p < 0.05 and ^∗∗p < 0.01.

FIGURE 5

Effect of FA on the popA expression in planta. Gray bars, DMSO control; dark bars, FA treatment. Cells suspension of RK5050 (OE1-1 popA-lacZYA) with FA supplementation or DMSO control was infiltrated into tobacco leaves. Leaf disks were punched every 6 hpi and bacterial cells was collected for β-galactosidase assay. Each assay included at least three samples from different plants and mean values of at least four independent trails were averaged and presented in RLU cell^-1 with SD (error bars).

FIGURE 6

Effect of FA on HR elicitation of R. solanacearum in tobacco leaves. (A) Bacterial suspension with FA supplementation, (B) bacterial suspension with DMSO control, (C) FA solution, (D) DMSO control. Approximately, 50 μl of RS1002 bacterial suspension at 10⁸ cfu ml^-1 was infiltrated into leaf mesophyll tissue with a blunt-end syringe. Pictures of tobacco leaves were taken at 16, 20, and 24 hpi. Each trail included at least seven plants and three independent trails were performed. Presented results were from approximately 20% (5 from total 21) tested tobacco leaves in which development of necrotic lesions was slightly faster than the that of control. Whereas no difference was observed in HR elicitation between FA treatment and the DMSO control in others 16 tested tobacco leaves.

FIGURE 7

Effect of FA on infection process of R. solanacearum. (A) By soil-soak inoculation on humus soil cultivating tomato plants, (B) by root sipping inoculation on hydroponic cultivating tomato plants. Bacterial suspension was poured onto the humus soil or hydroponic medium at a final concentration of or 10⁷ cfu g^-1 of soil or 10⁷ cfu ml^-1 of medium. Opened circles, DMSO control; opened triangles, FA treatment. Plants were inspected daily for wilt symptoms, and scored on a disease index scale from 0 to 4 (0, no wilting; 1, 1–25% wilting; 2, 26–50% wilting; 3, 51–75% wilting; and 4, 76–100% wilted or dead). Each trail included at least 12 plants and mean values of at least three independent trails were averaged and presented with SD (error bars). Statistical significance between FA treatment and the control was assessed using a post hoc Dunnett test following ANOVA. Significance level, ^∗p < 0.05 and ^∗∗p < 0.01.