Accumulation of the hormone abscisic acid (ABA) at the infection site of the fungus Cercospora beticola supports the role of ABA as a repressor of plant defence in sugar beet

Abstract

Inducible plant defence responses in sugar beet (Beta vulgaris L.) leaves are repressed during the early phase of infection by the fungus Cercospora beticola. In this report, we show that the concentration of the plant hormone abscisic acid (ABA) increases in sugar beet leaves during C. beticola infection. After an initial burst of ABA induced by inoculation of the fungus, elevated ABA concentrations were detected during the fungal penetration and colonization phases 3-9 days after inoculation. Fifteen days after inoculation, with visible onset of the necrotic phase of infection, the strongly elevated ABA concentrations in infected leaves were at levels similar to drought-stressed plants. A synthetic promoter composed of four copies of the ABA-responsive element (ABRE) A2 and the coupling element CE3 of the ABA-inducible barley gene HVA1 was strongly induced by ABA and C. beticola infection in transgenic sugar beet leaves. Analysis of the spatial pattern of promoter activity revealed that the ABA-inducible promoter was locally activated at the fungal infection sites. Furthermore, expression of the basic leucine zipper transcription factor AREB1 was induced by drought stress and fungal infection in the sugar beet. Application of ABA reduced the promoter activity of the phenylalanine ammonia lyase (BvPAL) gene, and this effect was observed with the -34 to +248 BvPAL promoter region. This region is equivalent to the core promoter, which is necessary for the suppression of BvPAL expression by C. beticola, as recently shown. These data indicate that ABA accumulation and activation of the ABA-dependent signalling cascade are the primary cause of suppression of BvPAL expression during infection of sugar beet leaves.

Figure 1

Accumulation of ABA during the biotrophic period of Cercospora beticola infection in sugar beet leaves. Determination of ABA in C. beticola infected and mock‐inoculated sugar beet leaves by competitive ELISA. The solid black squares represent the infected samples and the white circles the uninfected samples. Sugar beet plants were infected with 40 000 mycelium fragments and spores/mL of C. beticola in the greenhouse and then analysed immediately (0 dpi = days post inoculation) and at specific time points up until 9 dpi, during the biotrophic period of infection. The fungal development on sugar beet leaves is shown. The data shown are the means of six replicates from two mixed samples of leaves, and each mixed sample consisted of six middle‐sized leaves taken from six plants. The bars indicate the standard deviation. The asterisk indicates significant difference compared with the non‐infected control at the same time point (P < 0.05). The statistical analysis was performed with a two‐sided t‐test.

Figure 2

Accumulation of ABA and BvAREB1 transcripts during the necrotic period of Cercospora beticola infection and in leaves of drought‐stressed sugar beet. (A) Accumulation of the plant hormone ABA during the necrotic period of C. beticola infection. ABA concentrations in leaves without symptoms and leaves with slight, moderate and strong leaf spot symptoms were determined by ELISA at 15 dpi. The data shown are the means of six replicates. The standard deviation is shown by bars. The asterisk indicates significant difference compared with the uninfected control at the same time point (P < 0.05). The statistical analysis was performed with a two‐sided t‐test. (B) Accumulation of the plant hormone ABA during wilting of sugar beet leaves caused by water stress in the greenhouse. ABA levels were measured in leaves of water‐stressed plants and well‐watered control plants by ELISA 0, 24, 30, 72, 96 and 102 h after stopping the water supply. The data shown are the means of six replicates. The bars represent the standard deviation. The asterisk indicates significant difference from the control at the same point in time (P < 0.05). The statistical analysis was performed with a two‐sided t‐test. (C) Accumulation of BvAREB1 transcripts in sugar beet leaves with moderate and strong disease symptoms. BvAREB1 expression in the samples, previously analysed for ABA, was determined by semi‐quantitative RT‐PCR. Expression was normalized against levels of GAPDH expression to confirm equal cDNA amounts, and BvPR1 was used as a marker for defence gene activation. (D) Expression of the transcription factor BvAREB1 is induced in drought‐stressed plants 72, 96 and 102 h after stopping the water supply as shown by RNA blot analysis. No difference between control plants (–) and stressed plants (+) was detectable at the beginning of the experiment and after 30 h. BvAREB1 transcripts are marked by arrows. Equal loading of total RNA was confirmed by ethidium bromide staining (data not shown).

Figure 3

The synthetic promoter 4x(CE3‐A2) is responsive to exogenous ABA and Cercopora beticola infection in sugar beet leaves. (A) Schematic representation of the promoter reporter gene fusions of the 4x(CE3‐A2)‐LUC and 4x(A3‐CE1)‐LUC constructs. Four copies of the barley CE3‐A2 or A3‐CE1 cis‐elements are upstream of the 35S minimal promoter and the Photinus pyralis luciferase gene. The nucleotide sequences of the CE3‐A2 and A3‐CE1 elements are shown below each construct. (B) ABA induction of the synthetic promoter 4x(CE3‐A2) as shown by a transient assay. The reporter constructs 4x(CE3‐A2)‐LUC, 4x(A3‐CE1)‐LUC or the promoterless plasmid MS23‐LUC‐m3 (vector) were co‐transformed with the p70SRUC normalization vector into leaf discs by particle bombardment. Leaf discs were sprayed either with 100 µm cis/trans ABA or 5% DMSO as a control. (C) Transient ABA induction of the 4x(CE3‐A2) promoter in leaves of transgenic sugar beets. Two reporter gene lines (PR101/6 and PR101/35), which harbour the synthetic 4x(CE3‐A2) promoter upstream of the luciferase gene, and the non‐transgenic cultivar 3DC4156 (control) were sprayed with 100 µm cis/trans ABA or 5% DMSO. Specific luciferase activity (K × RLU/mg tissue) was determined 1, 24, 48, 72 and 96 h after hormone application. The average value of four replicates per treatment and the ABA inducibility of the promoter at each time point are given. The experiment was repeated twice with similar results. White columns (5% DMSO), black columns (100 µm ABA in 5% DMSO). (D) Induction of the 4x(CE3‐A2) promoter by Cercospora beticola infection in sugar beet leaves. The reporter gene lines, PR101/6 and PR101/35, and the non‐transgenic cultivar 3DC4156 were infected with C. beticola. Specific luciferase activity (K × RLU/mg tissue) was determined daily after infection. The average value of four replicates per treatment and the fungal induction of the promoter per time point are given. The experiment was repeated twice with similar results. White columns (non‐infected), black columns (infected).

Figure 4

Histochemical localization of luciferase activity in ABA‐treated and Cercospora beticola infected sugar beet leaves. The left column (A, C, E) shows specimens under dark field conditions detected by a CCD camera after application of luciferin; the right column (B, D, F) contains the same specimens observed with bright field optics. (A, B) Reporter gene line PR101/11, expressing the synthetic promoter 4x(CE3‐A2) upstream of the luciferase gene, and the non‐transgenic control 48 h after application of 100 µm cis/trans ABA. A PR101/11 plant in the absence of ABA treatment is shown as a control. (C, D) Local induction of the 4x(CE3‐A2) promoter activity in PR101/11 around the lesions caused by C. beticola infection in the in‐vitro assay at 8 dpi. Luciferase activity was absent in the leaf disc of the uninfected PR101/11 line (top plant). (E, F) Local induction of 4x(CE3‐A2) promoter activity around a C. beticola lesion observed in two leaf discs (1, 2) from PR101/11. Reporter gene activity was not detectable around the lesion of a non‐transgenic plant (3) or in the leaf disc of a uninfected transgenic PR101/11 plant (4). The plants were infected in the greenhouse and leaf discs were stained for luciferase activity.

Figure 5

ABA reduces the activity of the BvPAL minimal promoter. (A) Schematic representation of the ‐34‐BvPAL‐LUC reporter gene construct with the BvPAL minimal promoter and the 5′‐UTR from position +1 to +147. Stippled box = UTR, hatched box = coding region of BvPAL. (B) Reduction of the activity of the BvPAL minimal promoter by ABA in a transient assay. Normalized reporter gene activity of ‐34‐BvPAL‐LUC in ABA‐treated and untreated leaf discs of sugar beet. The p70SRUC vector was co‐transformed with the BvPAL reporter gene vector to control for differences in transformation efficiency.