Cytokinins mediate resistance against Pseudomonas syringae in tobacco through increased antimicrobial phytoalexin synthesis independent of salicylic acid signaling

Abstract

Cytokinins are phytohormones that are involved in various regulatory processes throughout plant development, but they are also produced by pathogens and known to modulate plant immunity. A novel transgenic approach enabling autoregulated cytokinin synthesis in response to pathogen infection showed that cytokinins mediate enhanced resistance against the virulent hemibiotrophic pathogen Pseudomonas syringae pv tabaci. This was confirmed by two additional independent transgenic approaches to increase endogenous cytokinin production and by exogenous supply of adenine- and phenylurea-derived cytokinins. The cytokinin-mediated resistance strongly correlated with an increased level of bactericidal activities and up-regulated synthesis of the two major antimicrobial phytoalexins in tobacco (Nicotiana tabacum), scopoletin and capsidiol. The key role of these phytoalexins in the underlying mechanism was functionally proven by the finding that scopoletin and capsidiol substitute in planta for the cytokinin signal: phytoalexin pretreatment increased resistance against P. syringae. In contrast to a cytokinin defense mechanism in Arabidopsis (Arabidopsis thaliana) based on salicylic acid-dependent transcriptional control, the cytokinin-mediated resistance in tobacco is essentially independent from salicylic acid and differs in pathogen specificity. It is also independent of jasmonate levels, reactive oxygen species, and high sugar resistance. The novel function of cytokinins in the primary defense response of solanaceous plant species is rather mediated through a high phytoalexin-pathogen ratio in the early phase of infection, which efficiently restricts pathogen growth. The implications of this mechanism for the coevolution of host plants and cytokinin-producing pathogens and the practical application in agriculture are discussed.

Figure 1.

Accumulation of CKs by overexpressing ipt causes resistance against PstT. A, Experimental scheme for transient 4xJERE:ipt expression in tobacco leaves followed by PstT infection. Tobacco leaves were preinfiltrated with A. tumefaciens containing [At(ipt); blue line] or without [At(0); black line] the 4xJERE:ipt construct followed by a second infiltration with either PstT (Pst; red line) or a mock infiltration with MgCl₂ (gray line) after 24 h. Numbers refer to time points of sampling for CK determination (Supplemental Table S1), JA and SA (Supplemental Table S2), free sugar (Supplemental Tables S3 and S4), antimicrobial activities (Table II), or phytoalexins (Table III). B, RNA-blot analysis of tobacco tissue transiently transformed with 4xJERE:ipt. A 28S rRNA loading control is shown at the bottom. C, Effect of transient 4xJERE:ipt expression and subsequent PstT infection on symptom development and green island formation. Tobacco leaves were preinfiltrated with A. tumefaciens with (ipt) or without (0) the 4xJERE:ipt construct followed by a second infiltration of PstT (Pst) or mock infiltration (0). The infiltrated areas are marked by circles. Photographs were taken at the indicated times (dpi). D, Effect of senescence-induced ipt expression in SAG12:ipt plants and subsequent PstT (red) infection on symptom development (left) in comparison with the wild type (right). E, Effect of tetracycline-induced ipt expression in TET:ipt plants and subsequent PstT infection on symptom development. Leaves of the transgenic TET:ipt tobacco line were preinfiltrated with tetracycline (TET; yellow) or water (0; gray) followed by a second infiltration with PstT (Pst; red). F, Effect of transient 4xJERE:ipt expression on the proliferation of PstT. Tobacco leaves were preinfiltrated with A. tumefaciens with (ipt) or without (0) the 4xJERE:ipt construct followed by a second infiltration with PstT. Bacteria were reisolated from leaf discs, plated, and quantified. Mean values ± sd of three independent replicates are shown.

Figure 2.

Exogenously supplied CKs enhance resistance against PstT. A, Experimental scheme of exogenous applications of CKs by petiole feeding (green bars) and dipping (yellow bar) prior to infection with PstT (red arrows). The petioles from both CK-treated and control leaves were kept in water for symptom development. B, Effect of feeding varying kinetin concentrations (1–18 μm) 24 h prior to PstT infection on symptom development at 7 dpi. C, Effect of short-pulse feeding (0.5–4 h) with 10 μm kinetin prior to PstT infection on symptom development at 13 dpi. D, Comparison of efficiency to induce resistance (12 dpi) against PstT for different adenine-derived CKs applied in 10 μm concentration at 24 h prior to infection. BAP, 6-Benzyladenine. E, Dose-dependent (2 fm to 200 nm) ability of phenylurea-derived CK TDZ to induce resistance against PstT (6 dpi) by petiole feeding. F, Induction of resistance against PstT (11 dpi) by dipping leaf halves for 60 s (left) and 90 s (right) in 140 μm kinetin 24 h before infection. Leaves were infiltrated with PstT at three sites per leaf half (B–F).

Figure 3.

Exogenously supplied CKs mediate resistance before as well as after PstT infection. A, Experimental scheme of varying starting times of the 24-h kinetin pulse (10 μm) relative to infection with PstT (24 h before infection [hbi] to 48 hpi). B, Effect of varying starting times of the 24-h kinetin pulse (10 μm) relative to infection with PstT (24 hbi to 48 hpi) on symptom development at 5 dpi. C, Experimental scheme for analysis of the continuance of CK-induced resistance with infections at different time points after CK feeding. D, CK-induced resistance effect (12 dpi) in leaves infected with PstT at the end of CK application (0 dpc) and several days after CK pulse (2–7 dpc) in comparison with the water control. Leaves were infiltrated at three sites with PstT (left) and 10 mm MgCl₂ (right; B and D).

Figure 4.

CKs induce resistance independent of SA, JA, ROS, and extracellular invertase activity. A, Effect of a 24-h kinetin application (10 μm) before PstT infection on symptom development in nahG-overexpressing tobacco leaves in comparison with the control at 8 dpi. Leaves were infiltrated at two sites with PstT (left) and 10 mm MgCl₂ (right). B, Effect of transient 4xJERE:ipt expression and PstT infection on H₂O₂ formation. Tobacco leaves were preinfiltrated with A. tumefaciens with (ipt) or without (0) the 4xJERE:ipt construct followed by a second infiltration with PstT (Pst). Leaves were stained with DAB to visualize H₂O₂ formation at 24 h after preinfiltration of A. tumefaciens (0 hpi; left), 24 h after infiltration of PstT (24 hpi; middle), and at the time of symptom development 168 h after PstT infiltration (168 hpi; right). C, Effect of CIN1 expression in TET:CIN1 tobacco leaves induced by a 24-h feeding of 1 mg L⁻¹ TET in comparison with a 24-h application of 10 μm kinetin prior to infection and water control treatment on symptom development at 10 dpi. Leaves were infiltrated with PstT at two sites per leaf half.

Figure 5.

CK-induced phytoalexin production causes pathogen resistance. A, Experimental data for time-course analysis of scopoletin production (blue line with circles; nmol g⁻¹ fresh weight [fw]) in relation to bacterial growth (red line with squares; cfu mL⁻¹) in PstT-infected leaves pretreated with CKs. Note the different scale for the scopoletin levels compared with B. B, Experimental data for scopoletin production and bacterial growth in PstT-infected leaves (control). C, Model for CK-mediated phytoalexin production based on A. CK feeding mediates increased phytoalexin levels, resulting in a high phytoalexin-bacteria ratio, which restricts bacterial growth in the early infection phase. In the late infection phase, bacterial growth is abolished, probably due to additional defense mechanisms triggered by the bacterial infection. D, Model for pathogen-induced (control) phytoalexin production based on B. Phytoalexin production is triggered by the bacterial infection, but the increase occurs after the onset of bacterial growth. This results in a low phytoalexin-bacteria ratio throughout the infection phase and consequently unrestricted bacterial growth. E, RNA-blot analyses of tobacco tissue transiently transformed with 4xJERE:ipt for expression levels of EAS, C4H, and TOGT. A 28S rRNA loading control is shown at the bottom. F, Phytoalexin infiltration confers pathogen resistance, as evident by reduced symptom development 6 dpi. W38 leaves were infiltrated with different concentrations of capsidiol and scopoletin (200–400 μm; yellow) or controls (gray) simultaneous with PstT bacteria (10⁷ cfu mL⁻¹; red).