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Keywords:

  • BABA-induced resistance;
  • necrotrophic pathogens;
  • abscisic acid;
  • jasmonic acid;
  • salicylic acid;
  • camalexin

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

The non-protein amino acid β-amino-butyric acid (BABA) protects plants against a wide range of pathogens. We have examined the effectiveness and mode of action of BABA on resistance against two necrotrophic pathogens. Treatment of Arabidopsis with BABA induced resistance against Alternaria brassicicola and Plectosphaerella cucumerina to a similar level by jasmonic acid (JA). Conversely, treatment with benzothiadiazole (BTH), a functional analogue of salicylic acid (SA), had no significant effect on the resistance against both pathogens. BABA-induced resistance against A. brassicicola and P. cucumerina was unaffected in the JA-insensitive mutant coi1-1 and the camalexin-deficient mutant pad3-1. Moreover, the expression of BABA-induced resistance was not associated with enhanced accumulation of camalexin or enhanced transcription of the JA-inducible PDF1.2 gene. The expression of BABA-induced resistance against P. cucumerina was unaffected in mutants impaired in ethylene (ET) and SA signalling, but was blocked in the abscisic acid (ABA)-deficient mutant aba1-5, the ABA-insensitive mutant abi4-1 and the callose-deficient mutant pmr4-1. Upon infection by both pathogens, BABA-treated plants showed an earlier and more pronounced accumulation of callose. Treatment with the callose-inhibitor 2-deoxy-D-glucose (2-DDG) reversed the BABA-induced resistance against A. brassicicola. Furthermore, primed callose deposition was absent in BABA-treated abi4-1 and pmr4-1 plants upon infection by P. cucumerina. Although the expression of BABA-induced resistance was not associated with enhanced transcription of the ABA-inducible RAB18 gene, application of ABA mimicked the effect of BABA on the level of callose accumulation and resistance. Hence, BABA-induced resistance against necrotrophic pathogens is based on primed callose accumulation, which is controlled by an ABA-dependent defence pathway.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

The success of a plant to combat microbial pathogens depends on speed. Early recognition of the pathogen is critical if the plant is to mobilise the available biochemical and structural defences to effectively halt the invading pathogen. If the plant fails to do so, the appropriate defences are activated too late, and the pathogen starts colonising the plant tissue. During this stage of infection, the plant responds by activating defences around the sites of pathogen invasion. This so-called basal resistance contributes to slow down the colonisation by the pathogen, but it is too weak and too late to prevent disease.

Biotrophic pathogens, which parasitise living plant cells, are controlled by other basal defence mechanisms as compared to necrotrophic pathogens, which immediately kill host cells to metabolise their contents. In Arabidopsis, activation of salicylic acid (SA)-dependent defence mechanisms enhances the level of basal resistance against the biotrophic pathogens Hyaloperonospora parasitica and turnip crinkle virus (Delaney et al., 1994; Kachroo et al., 2000; Uknes et al., 1993), whereas jasmonic acid (JA)- and ethylene (ET)-dependent defences yield no resistance against these pathogens (Thomma et al., 1998). Conversely, JA- and ET-dependent defence mechanisms significantly contribute to the level of basal resistance against necrotrophic pathogens, such as Botrytis cinerea, Alternaria brassicicola and Plectosphaerella cucumerina (Berrocal-Lobo et al., 2002; Thomma et al., 1998, 1999a). Furthermore, the Arabidopsis mutant pad3-1, which is impaired in the biosynthesis of the phytoalexin camalexin, shows reduced levels of basal resistance against the necrotrophic fungi A. brassicicola and B. cinerea (Ferrari et al., 2003; Thomma et al., 1999b), but develops wild-type levels of resistance against the biotrophic pathogens Pseudomonas syringae and H. parasitica (Glazebrook and Ausubel, 1994; Van Wees et al., 2003). Thus, the efficiency of the plant's basal resistance response depends on the challenging pathogen.

Besides basal resistance that is expressed locally upon primary pathogen attack, plants are also capable of enhancing their level of resistance against future pathogen attack. This phenomenon is known as induced resistance and can be triggered by a variety of biotic and abiotic stimuli. The classic example of induced resistance is often referred to as systemic acquired resistance (SAR), and occurs in distal plant parts upon localised infection by a necrosis-inducing pathogen (Ryals et al., 1996). The expression of pathogen-induced SAR depends on endogenous accumulation of SA and is marked by the transcriptional activation of SA-inducible genes encoding pathogenesis-related (PR) proteins (Gaffney et al., 1993; Lawton et al., 1994; Nawrath and Métraux, 1999). Apart from pathogen-induced SAR, selected strains of non-pathogenic rhizobacteria can induce systemic resistance as well. This so-called rhizobacteria-mediated induced systemic resistance (ISR) is not associated with transcriptional activation of genes encoding PR proteins (Pieterse et al., 1996; Van Wees et al., 1997), and functions independently of SA, but requires intact responsiveness to the plant hormones JA and ET (Pieterse et al., 1998). Besides these differences in signalling pathway, ISR and SAR also differ in the spectrum of pathogens against which they yield protection. Whereas SAR is mostly effective against biotrophic pathogens that are resisted by SA-dependent basal resistance, ISR predominantly yields protection against necrotrophic pathogens that are sensitive to JA- and ET-dependent basal resistance (Ton et al., 2002).

Apart from SAR and ISR, treatment with the non-protein amino acid β-amino-butyric acid (BABA) has demonstrated to activate an induced resistance response as well. Although BABA is found rarely as a natural occurring compound in plants, it is a potent inducer of broad-spectrum disease resistance in different plant species (Cohen, 2002; Jakab et al., 2001). Non-protein amino acids, such as BABA and its isomer γ-amino-butyric acid (GABA), have known biological effects in animals and plants. For instance, GABA functions as an inhibitory neurotransmitter in the central nervous system of animals (Waagepetersen et al., 1999), whereas BABA can act as a partial agonist of the glycine receptor (Schmieden and Betz, 1995). In plants, GABA is produced in response to stress (Shelp et al., 1999), although its ability to induce resistance against plant diseases remains controversial (Cohen, 2002; Shelp et al., 1999). In contrast, the broad-spectrum protective effect of BABA against numerous diseases and abiotic stresses is well documented in plants (Jakab et al., 2001). Research in Arabidopsis revealed that the signalling pathway controlling BABA-induced resistance is specific for the challenging pathogen. BABA-induced resistance against the bacterial pathogen P. syringae pv. tomato DC3000 and the fungal pathogen B. cinerea resembles pathogen-induced SAR in that it depends on endogenous accumulation of SA and an intact NPR1 protein (Zimmerli et al., 2000, 2001). Although treatment with BABA alone did not induce the expression of SA- and JA-/ET-inducible PR genes, plants treated with BABA showed a faster and stronger transcription of the SA-inducible PR-1 gene upon infection with P. syringae, similar to that observed during the expression of biologically induced SAR (Cameron et al., 1999; Kohler et al., 2002). However, BABA-induced resistance against the oomycete pathogen H. parasitica was fully functional in Arabidopsis genotypes impaired in SAR and ISR signalling (Zimmerli et al., 2000). This indicates that BABA can activate resistance through a yet unidentified signalling pathway.

A common characteristic of the various induced resistance phenomena is that they are all associated with an enhanced capacity to express cellular defence responses, which are induced specifically upon attack by a pathogen. By analogy with a phenotypically similar phenomenon in mammalian monocytes and macrophages, the capacity to express a faster and stronger basal defence response upon pathogen infection is called priming (Conrath et al., 2002). In Arabidopsis, the enhanced resistance by BABA is based on various priming mechanisms. BABA-induced resistance against P. syringae and B. cinerea was found to correlate with primed transcription of the SA-inducible PR-1 gene in wild-type plants, whereas NahG and npr1 plants failed to express BABA-induced resistance against these pathogens (Zimmerli et al., 2000, 2001). This clearly demonstrates that priming for SA-inducible defences is the predominant mechanism by which BABA protects against P. syringae and B. cinerea. In contrast, priming for SA-inducible defences cannot explain the protection against H. parasitica, as SA non-accumulating NahG plants and SA-insensitive npr1 plants were fully capable of expressing BABA-induced resistance against this pathogen (Zimmerli et al., 2000). Interestingly, the expression of the BABA-induced resistance against H. parasitica correlated with an earlier and stronger formation of callose-rich papillae around the growing hyphae of the pathogen (Zimmerli et al., 2000), suggesting that BABA-induced resistance against H. parasitica is based on primed callose deposition. However, conclusive evidence that enhanced callose deposition was the actual mechanism behind this BABA-induced resistance was not provided. Recently, we found that BABA is also effective against osmotic stress (G. Jakab and J. Ton, unpublished data). This BABA-induced protection against salt and drought stresses was functional in Arabidopsis genotypes impaired in SA, JA or ET signalling, but was blocked in Arabidopsis mutants impaired in the production or sensitivity to the abiotic stress hormone abscisic acid (ABA). Additionally, BABA-treated wild-type plants expressed ABA-inducible marker genes at lower salt concentrations than non-induced plants, indicating that BABA-induced protection against osmotic stress is achieved through priming of ABA-dependent defence mechanisms. Hence, BABA triggers a primed state in the plant that enables a more efficient activation of SA-dependent defence mechanisms, callose deposition or ABA-dependent defence mechanisms.

To gain more insight into the SA-independent component of BABA-induced resistance, we investigated the effectiveness and mode of action of BABA against two necrotrophic pathogens that are relatively insensitive to SA-induced resistance. Here, we show that the expression of BABA-induced resistance against A. brassicicola and P. cucumerina is not based on SA-, JA- or ET-dependent defences, or on enhanced accumulation of camalexin. Instead, BABA-induced resistance against these necrotrophs is primarily based on a primed accumulation of callose. In addition, we provide evidence that this primed callose response is regulated by an ABA-dependent signalling pathway.

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

Comparison between BABA-induced resistance and benzothiadiazole (BTH)- and JA-induced resistance against A. brassicicola and P. cucumerina

Wild-type Arabidopsis exhibits full resistance against the necrotrophic fungus A. brassicicola and is therefore unsuitable for studying induced resistance against the fungus. In contrast, the camalexin-deficient mutant pad3-1 as well as the JA-insensitive mutant coi1-1 are susceptible to A. brassicicola (Thomma et al., 1998, 1999b). To determine whether BABA-induced resistance is effective against A. brassicicola, 6-week-old pad3-1 plants were treated with either water or BABA, and subsequently challenged with A. brassicicola. Compared to water-treated control plants, soil drench treatment with 150 µm BABA resulted in a statistically significant reduction of lesion size at 4 days after challenge inoculation (Figures 1a and 2a). The level of BABA-induced resistance was comparable to the level of resistance induced by soil drench treatment with 100 µm JA. In contrast, treatment with 300 µm of SA analogue BTH yielded no protection against A. brassicicola, even though this amount of BTH was enough to activate SA-inducible PR-1 transcription (data not shown). To determine the effectiveness of BABA-induced resistance against the necrotrophic fungus P. cucumerina, 5-week-old Col-0 plants were treated with 150 µm BABA, and were subsequently challenged with P. cucumerina spores. Compared to water-treated control plants, treatment with BABA induced a statistically significant reduction in lesion size at 6 days after challenge inoculation (Figures 1b and 2b). Also in this interaction, the level of BABA-induced resistance was comparable to that of JA-induced resistance. Treatment with 300 µm BTH resulted in a relatively weak reduction in lesion size compared to control plants, which was not statistically significant (Figure 2b). Apparently, SA-inducible resistance is considerably less effective than BABA- and JA-induced resistance against P. cucumerina.

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Figure 1. Lesion development on Arabidopsis leaves infected with A. brassicicola and P. cucumerina.

(a) A. brassicicola: 6-week-old pad3-1 plants were soil-drenched with water or 150 µm BABA at 2 days prior to challenge inoculation with 6-µl droplets containing 2 × 106 spores ml−1. Pictures were taken at 4 days after inoculation.

(b) P. cucumerina: 5-week-old Col plants were soil-drenched with water or 150 µm BABA at 2 days prior to challenge inoculation with 6-µl droplets containing 5 × 106 spores ml−1. Pictures were taken at 6 days after inoculation.

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Figure 2. Quantification of BTH-, BABA- and JA-induced resistance against A. brassicicola and P. cucumerina.

(a) A. brassicicola: 6-week-old pad3-1 plants were soil-drenched with water, 300 µm BTH, 150 µm BABA or 100 µm JA. At 2 days after chemical treatment, 6–8 leaves per plant were challenged by applying 6-µl droplets containing 2 × 106 spores ml−1. Average lesion diameters for 20–30 plants per treatment were determined at 4 days after inoculation.

(b) P. cucumerina: 5-week-old Col plants were chemically treated as described for (a). Challenge inoculation was performed at 2 days after chemical treatment by applying 6-µl droplets containing 5 × 106 spores ml−1. Average lesion diameters were determined at 6 days after inoculation.

Values presented are means ± SEM. Different letters indicate statistically significant differences (Fisher's least significant difference (LSD) test; α = 0.05).

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The role of JA- and SA-dependent defences in BABA-induced resistance against A. brassicicola and P. cucumerina

Because treatment with BABA protected Arabidopsis to a similar level as treatment with JA (Figure 2), we examined the possibility that BABA-induced resistance against A. brassicicola and P. cucumerina is based on primed expression of JA-inducible defences. To this end, we tested the effectiveness of BABA in JA-insensitive coi1-1 plants. Treatment of this mutant with 150 µm BABA resulted in a statistically significant reduction in lesion size upon infection with A. brassicicola and P. cucumerina (Figure 3). This indicates that JA-dependent defences are not required for the expression of BABA-induced resistance against both pathogens. To further test the involvement of JA-dependent defence mechanisms, we examined the expression of the JA-inducible PDF1.2 gene in pad3-1 and Col plants upon infection with A. brassicicola or P. cucumerina, respectively. RNA gel blot analysis revealed that treatment with BABA alone did not induce transcription of the PDF1.2 marker gene (Figure 4). Moreover, pathogen-induced PDF1.2 transcription was not enhanced in BABA-treated plants compared to water-treated plants, indicating that BABA-induced resistance against A. brassicicola and P. cucumerina is not based on primed expression of JA-inducible defences. Hybridisation of the blots with a probe for the SA-inducible PR-1 gene revealed that BABA treatment caused an earlier and stronger induction of PR-1 upon infection by both pathogens, although treatment with BABA alone had no effect on the gene (Figure 4). It can thus be concluded that BABA primes for SA-dependent defence responses upon infection by both fungi.

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Figure 3. Quantification of BABA-induced resistance in different Arabidopsis genotypes against A. brassicicola (a) and P. cucumerina (b). For experimental details, see legend to Figure 2. Asterisks indicate statistically significant differences compared to water-treated control plants (Student's t-test; α = 0.05).

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Figure 4. RNA gel blot analysis of PDF1.2, PR-1 and RAB18 gene expression in water- and BABA-treated plants at different hours after infection with A. brassicicola (a) or P. cucumerina (b).

Six-week-old plants were soil-drenched with water or 150 µm BABA at 2 days before challenge inoculation with A. brassicicola or P. cucumerina. Induction of RAB18 gene expression was verified in 6-week-old Col plants at 2 days after treatment with 150 µm BABA, 150 mm NaCl or 80 µm ABA (c). Each time-point represents 20 leaves per treatment collected from four different plants. RNA blots were hybridised with PR-1, PDF1.2 or RAB18 probes. Ethidium bromide staining of the RNA gel (rRNA) was used to show equal loading. hpi, hours post-inoculation.

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The role of camalexin in BABA-induced resistance against A. brassicicola and P. cucumerina

To test whether camalexin plays a role in BABA-induced resistance against A. brassicicola and P. cucumerina, we quantified BABA-induced resistance in camalexin-deficient pad3-1 plants. Treatment with BABA induced normal levels of resistance against A. brassicicola and P. cucumerina (Figures 1a and 3), indicating that camalexin is not required for the induced resistance. Furthermore, quantification of camalexin levels revealed that BABA-treated coi1-1 and Col plants did not accumulate enhanced levels of camalexin upon infection with A. brassicicola or P. cucumerina, respectively (Figure 5). In contrast, water-treated control plants accumulated significantly more camalexin than BABA-treated plants. This clearly demonstrates that BABA-induced resistance against A. brassicicola and P. cucumerina is not based on primed accumulation of camalexin.

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Figure 5. Quantification of camalexin production in water- and BABA-treated coi1-1 (a) and Col (b) during infection with A. brassicicola and P. cucumerina, respectively.

Six-week-old plants were soil-drenched with water or 150 µm BABA at 2 days prior to challenge inoculation. Camalexin was extracted at different time-points after inoculation and quantified by luminescence/fluorescence spectrometry. Each time-point represents six infected leaves collected from three different plants. Camelexin quantities were expressed relative to the sample containing the highest emission value.

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BABA-induced resistance against P. cucumerina depends on an ABA-dependent signalling pathway

As described above, BABA-induced resistance against P. cucumerina is not affected in pad3-1 and coi1-1 plants. Similarly, ET-insensitive (ein2-1) plants, SA non-accumulating NahG plants and SA-insensitive npr1-1 plants were fully capable of expressing BABA-induced resistance against P. cucumerina (Figure 3b). Apparently, camalexin as well as SA-, JA- and ET-dependent defence mechanisms do not contribute to BABA-induced resistance against this pathogen. In contrast, the ABA-deficient mutant aba1-5 (Koornneef et al., 1982) and the ABA-insensitive mutant abi4-1 (Finkelstein, 1994), which are both in the background of accession Col-0, were completely blocked in their ability to express BABA-induced resistance against P. cucumerina (Figure 3b). This indicates that the expression of BABA-induced resistance against P. cucumerina requires intact ABA signalling. Quantification of BABA-induced resistance against A. brassicicola in ein2-1, npr1-1, NahG, aba1-5 and abi4-1 was not possible, because all these genotypes exhibited full resistance against the fungus (data not shown). To examine whether the expression of BABA-induced resistance against P. cucumerina and A. brassicicola is accompanied with enhanced production of ABA, we tested the expression of the ABA-inducible RAB18 gene upon infection by both pathogens. RNA blot analysis showed no enhanced transcription of the RAB18 gene in water- and BABA-treated plants upon infection by both pathogens (Figure 4a,b), whereas treatments with 150 mm NaCl or 80 µm ABA were both effective in inducing the expression of the RAB18 gene (Figure 4c). These findings suggest that the BABA-induced resistance is not mediated by major rises in ABA levels.

BABA-induced resistance against A. brassicicola is based on a primed accumulation of callose

To gain more insight into the nature of BABA-induced resistance against A. brassicicola, cytological observations were performed at the sites of infection. At 2 days after inoculation, approximately half of the spores had germinated on the leaves of pad3-1 plants. Although treatment with BABA had no obvious effect on the efficiency of spore germination, the length of the germ tubes was significantly reduced on leaves of BABA-treated plants (Figure 6a,b). Furthermore, germinating spores induced significantly more papillae in the epidermal cell layer of BABA-treated plants than in leaves of control plants (Figure 6c). Aniline blue staining revealed that these papillae contained callose (Figure 6d,e). Thus, the expression of BABA-induced resistance against A. brassicicola is associated with primed deposition of callose-rich papillae. To determine whether this primed callose response contributes to the BABA-induced resistance against the pathogen, the petioles of detached leaves of 5-week-old pad3-1 plants were placed in tubes containing 150 µm BABA with increasing concentrations of the callose inhibitor 2-deoxy-D-glucose (2-DDG; Jaffe and Leopold, 1984). After 24 h of incubation, the leaves were challenged with A. brassicicola spores. Callose accumulation was quantified microscopically by determining the percentage of callose-inducing spores at 2 days after inoculation (Figure 7a), and the level of induced resistance was quantified by determining average lesion diameters at 4 days after inoculation (Figure 7b). Leaves incubated in 150 µm BABA showed a 40% reduction in lesion size compared to water-incubated control leaves (Figure 7a). This enhanced resistance correlated with a threefold increase in the percentage of callose-inducing spores (Figure 7b). Co-application of BABA with increasing concentrations of 2-DDG reduced the percentage of callose-inducing spores. At 250 µm 2-DDG, the level of callose-inducing spores was comparable to that in leaves of non-induced plants (Figure 7a). The reduction in callose accumulation correlated with an increase in average lesion size. At a concentration of 250 µm 2-DDG, the resistance-inducing effect of BABA was completely abolished (Figure 7b). Thus, inhibition of callose deposition and possibly other associated responses by 2-DDG reduces BABA-induced resistance against A. brassicicola. Nevertheless, treatment with 2-DDG did not severely affect papillae formation (data not shown). This suggests that papillae can still occur in the absence of callose, but have no further contribution to the BABA-induced resistance.

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Figure 6. Tissue colonisation by A. brassicicola and P. cucumerina in water- and BABA-treated Arabidopsis at 2 days post-inoculation (dpi).

(a) A. brassicicola in water-treated pad3-1 plants (bar = 100 µm).

(b) A. brassicicola in BABA-treated pad3-1 plants (bar = 100 µm).

(c) Papillae formation (arrow) in an epidermal cell surrounded by germ tubes of A. brassicicola in BABA-treated pad3-1 (bar = 25 µm).

(d) A. brassicicola in water-treated pad3-1 plants (bar = 25 µm).

(e) Callose deposition (arrow) around an appressorium of A. brassicicola in BABA-treated pad3-1 (bar = 25 µm).

(f) P. cucumerina in water-treated Col plants (bar = 25 µm).

(g) Papillae formation (arrows) in an epidermal cell surrounded by germinating spores of P. cucumerina in BABA-treated Col plants (bar = 25 µm).

(h) P. cucumerina in water-treated Col plants (bar = 15 µm).

(i) Callose deposition (arrow) around germinating spores of P. cucumerina in BABA-treated Col plants at 2 dpi (bar = 15 µm).

Leaves were stained with lactophenol/trypan blue and analysed by light microscopy (a–c,f,g), or stained with calcofluor/aniline blue and analysed by epifluorecence microscopy (UV; d,e,h,i).

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Figure 7. Effect of the callose-inhibitor 2-DDG on the level of callose deposition and BABA-induced resistance against A. brassicicola.

Leaves of 5-week-old pad3-1 plants were detached and incubated for 24 h in Eppendorf tubes containing different concentration of BABA and 2-DDG. Leaves were inoculated with 2 × 106 spores ml−1.

(a) Callose was visualised by aniline blue staining and epifluorescence microscopy (UV) at 2 days post-inoculation (dpi). Data shown are means (±SEM; n = 8) of the percentage of callose-inducing spores per leaf.

(b) Resistance was quantified by measuring lesion diameters at 4 dpi. Data shown are means (±SEM; n = 15). Different letters indicate statistically significant differences (Fisher's LSD test; α = 0.05).

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BABA-induced resistance against P. cucumerina is based on ABA-dependent priming for callose accumulation

To assess the involvement of callose in BABA-induced resistance against P. cucumerina, cytological observations were performed at the sites of infection in water- and BABA-treated Col plants. As observed during the interaction with A. brassicicola, BABA-treated plants developed significantly more callose-containing papillae than non-induced control plants at 2 days after inoculation with P. cucumerina (Figure 6f–i). To determine whether this primed callose deposition is responsible for the BABA-induced resistance against P. cucumerina, we have determined the level of callose accumulation and BABA-induced resistance in water- and BABA-treated Col and pmr4-1 plants. The pmr4-1 mutant carries a mutation in the callose synthase gene AtGSL5 and fails to synthesise callose in response to pathogen infection and wounding (Jacobs et al., 2003; Nishimura et al., 2003). At different time points after inoculation, infected leaves were collected, stained with aniline blue and examined by UV fluorescence microscopy. Callose deposition was quantified by determining the relative number of yellow pixels on digital photographs that correspond to pathogen-induced callose. As shown in Figure 8, leaves of non-induced Col plants transiently increased callose deposition upon P. cucumerina infection. Treatment of Col plants with BABA resulted in an earlier and stronger deposition of callose, demonstrating the primed callose response (Figure 8a,b). In contrast, both water- and BABA-treated pmr4-1 plants failed to accumulate any detectable levels of callose upon infection by P. cucumerina (Figure 8a). Moreover, pmr4-1 plants failed to express BABA-induced resistance against the fungus (Figure 3b), indicating that callose is a critical factor in the establishment of BABA-induced resistance against P. cucumerina. As observed in response to treatment with 2-DDG, papillae formation was not severely affected in water- and BABA-treated pmr4-1 plants (data not shown). This indicates that papillae can still occur in the absence of callose, but have no further contribution to the BABA-induced resistance.

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Figure 8. Callose accumulation in water- and BABA-treated Col (a,b), pmr4-1 (a) and abi4-1 (b) upon infection by P. cucumerina.

Callose was visualised by aniline blue staining and epifluorescence microscopy (UV). Quantification was performed by determining the number of yellow pixels per million pixels corresponding to pathogen-induced callose on digital photographs of infected leaf areas. Data shown are means (±SEM; n = 8) of the relative number of yellow pixels per photograph.

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To further investigate the relationship between ABA signalling, callose deposition and BABA-induced resistance against P. cucumerina, we compared the BABA-induced callose response of Col plants to that of abi4-1 plants, which, like aba1-5 and pmr4-1, are impaired in BABA-induced resistance against the fungus (Figure 3b). Water-treated abi4-1 plants showed similar kinetics in callose accumulation as water-treated Col plants, indicating that this mutant, unlike pmr4-1, is not affected in pathogen-inducible callose formation (Figure 8b). However, BABA-treated abi4-1 plants failed to accumulate enhanced levels of callose upon pathogen infection. This indicates that the abi4-1 mutation affects BABA-induced resistance against P. cucumerina by blocking the primed callose deposition.

Application of ABA mimics the effect of BABA on the level of callose deposition and resistance against A. brassicicola and P. cucumerina

To further examine the involvement of ABA in callose-mediated resistance against A. brassicicola and P. cucumerina, we determined the effect of exogenous application of ABA on the level of callose deposition and pathogen resistance upon infection with A. brassicicola and P. cucumerina. Soil drench treatment with 80 µm ABA resulted in a fivefold increase in the number of callose-inducing spores at 2 days after challenge inoculation with A. brassicicola (Figure 9a). Similarly, the level of callose accumulation in ABA-treated plants following P. cucumerina infection was increased 10-fold as compared to water-treated control plants (Figure 9a). This enhanced callose production correlated with increased levels of pathogen resistance, as evidenced by a statistically significant reduction in lesion size at 4 and 6 days after inoculation with A. brassicicola and P. cucumerina, respectively (Figure 9b). The extent of the ABA-induced effects on callose and pathogen resistance was comparable with that observed after treatment with BABA. Apparently, activation of the ABA response primes for pathogen-inducible callose deposition, causing enhanced levels of resistance against A. brassicicola and P. cucumerina.

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Figure 9. Effect of exogenous application of BABA and ABA on the level of callose deposition (a) and resistance (b) after inoculation with A. brassicicola and P. cucumerina. Five-week-old pad3-1 or Col plants were soil-drenched with water, 150 µm BABA or 80 µm ABA at 2 days before challenge inoculation with A. brassicicola and P. cucumerina, respectively. For further experimental details, see legends to Figures 2 and 8. Different letters indicate statistically significant differences (Fisher's LSD test; α = 0.05).

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Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

In this study, we have investigated the effectiveness and mode of action of BABA-induced resistance against two necrotrophic pathogens. We have shown that treatment with BABA induces resistance against A. brassicicola and P. cucumerina, which are both unaffected by SA-induced defence mechanisms. This confirms the earlier notion that BABA-induced resistance can be a mechanistically different resistance response than SA-dependent SAR (Zimmerli et al., 2000). To elucidate the mechanisms behind the BABA-induced resistance against A. brassicicola and P. cucumerina, we examined the involvement of SA-, JA- and ET-dependent defences, camalexin accumulation and callose deposition.

Previously, we reported that BABA-induced resistance against the necrotrophic fungus B. cinerea is based on a primed expression of SA-dependent defence mechanisms (Zimmerli et al., 2001). This conclusion was based on the observation that SA non-accumulating NahG plants failed to express BABA-induced resistance against the fungus, and that BABA-treated wild-type plants showed primed expression of the SA-inducible marker gene PR-1. In this study, we found similar priming for PR-1 expression after infection by A. brassicicola and P. cucumerina (Figure 4). However, this primed defence reaction cannot explain the BABA-induced resistance against both pathogens. In the first place, treatment with the SA analogue BTH failed to induce significant levels of resistance against A. brassicicola and P. cucumerina (Figure 2), indicating that activation of SA-inducible defence mechanisms is ineffective against both pathogens. Secondly, BABA-induced resistance against P. cucumerina was fully functional in SA non-accumulating NahG plants and SA-insensitive npr1-1 plants (Figure 3b). Hence, BABA primes for SA-dependent resistance against A. brassicicola and P. cucumerina, but this primed defence expression is not effective against these pathogens, and can therefore not explain the BABA-induced resistance.

JA- and ET-dependent defences contribute to the level of basal resistance against A. brassicicola and P. cucumerina (Berrocal-Lobo et al., 2002; Thomma et al., 1998). Accordingly, we found enhanced susceptibility in the JA-insensitive mutant coi1-1 to A. brassicicola, and enhanced susceptibility to P. cucumerina in the ET-insensitive mutant ein2-1 (Figure 3). Despite the apparent function of JA and ET in basal resistance against A. brassicicola and P. cucumerina, the expression of BABA-induced resistance against both pathogens was not affected by the coi1-1 and ein2-1 mutations, respectively (Figure 3). Moreover, transcriptional analysis of the JA- and ET-inducible PDF1.2 gene did not reveal primed activity of JA- and ET-dependent defence pathway in BABA-treated plants (Figure 4). It can thus be concluded that the protection by BABA against A. brassicicola and P. cucumerina is not based on potentiation for JA- and ET-inducible defences.

Another factor involved in Arabidopsis resistance against necrotrophic fungi is the phytoalexin camalexin. Characterisation of the camalexin-deficient mutant pad3-1 showed that loss of camalexin confers enhanced susceptibility to A. brassicicola (Thomma et al. 1999b) and B. cinerea (Ferrari et al., 2003). In support of these findings, we found that the level of basal resistance against A. brassicicola and P. cucumerina is severely affected by the pad3-1 mutation (Figure 3). However, expression of BABA-induced resistance against both pathogens was unaffected in this mutant (Figure 3), and there was no primed camalexin production in BABA-treated coi1-1 and wild-type plants upon infection by A. brassicicola and P. cucumerina, respectively (Figure 5). Hence, camalexin does not contribute to the BABA-induced resistance against both necrotrophic pathogens. Surprisingly, non-induced control plants accumulated significantly more camalexin than BABA-treated plants upon pathogen infection. This difference can be explained by the higher level of disease incidence in non-induced plants compared to the more resistant BABA-treated plants. Because the level of pathogen colonisation is higher in the more susceptible non-induced plants, there are more cells that are directly attacked by the invading fungus. Consequently, there are more cells producing camalexin as part of the basal resistance response. The reduced camalexin production in BABA-treated plants also indicates that pathogen colonisation in BABA-treated plants is slowed down before the production of camalexin is triggered. This suggests that the mechanism behind BABA-induced resistance acts during an earlier stage of infection than camalexin.

Treatment with BABA resulted in an earlier and more pronounced accumulation of callose upon infection with A. brassicicola and P. cucumerina(Figures 6–9). Inhibition of this primed callose response by co-application of the callose inhibitor 2-DDG inhibited the BABA-induced resistance against A. brassicicola (Figure 7). Similarly, loss of callose accumulation in the pmr4-1 mutant coincided with its inability to express BABA-induced resistance against P. cucumerina (Figures 3b and 8b). Furthermore, the BABA-mediated priming for callose during P. cucumerina infection was absent in the abi4-1 mutant (Figure 8b), which concurrently lacked expression of BABA-induced resistance against this pathogen (Figure 3b). Together, these data strongly suggest that primed callose deposition is critical for the BABA-induced resistance against A. brassicicola and P. cucumerina.

Callose is an amorphous high-molecular weight β-1,3-glucan that is deposited in the form of papillae upon infection by fungal and oomycetous pathogens. It is thought to act as a physical barrier against fungal colonisation of the intercellular space. Two research groups have independently found that mutations in the callose synthase gene AtGSL5 of Arabidopsis, such as pmr4-1, paradoxically confer enhanced resistance against powdery mildew fungi (Jacobs et al., 2003; Nishimura et al., 2003). Blocking the SA-dependent defence pathway in pmr4-1 by introducing the NahG gene, or pad4 or npr1 mutations, reversed the enhanced resistance against powdery mildew, but did not restore callose deposition. This indicates that the enhanced resistance of pmr4-1 is caused by a hyperactive SA response (Nishimura et al., 2003). Such enhanced responsiveness to SA might be caused by a disturbance of cell wall homeostasis that could lead to a conditioning effect on SA-dependent resistance. Alternatively, the hyperactive SA response of pmr4-1 could suggest that callose or callose synthase negatively regulates the SA pathway. Nishimura et al. (2003) hypothesised that rapid callose deposition during the early stages of infection may repress later defence responses that may potentially be harmful for the plant. If a pathogen has evolved mechanisms to overcome the callose defence barrier, conditions become favourable for further colonisation because of repression of SA-dependent defences by callose. However, A. brassicicola and P. cucumerina would not benefit from a repressed SA response, as they are not affected by SA-inducible resistance (Figure 2). Moreover, A. brassicicola and P. cucumerina apparently lack mechanisms to circumvent callose deposition as they are effectively halted by BABA-mediated callose deposition. Interestingly, BABA treatment has been reported to be ineffective against powdery mildew pathogens (Cohen, 2002; L. Zimmerli, personal communication). Therefore, one may envisage that the insensitivity of powdery mildew pathogens to BABA-induced defences is because of their ability to overcome the callose defence barrier.

Treatment with BABA failed to induce resistance against P. cucumerina in the ABA-deficient mutant aba1-5 and the ABA-insensitive mutant abi4-1 (Figure 3b). Furthermore, we observed that the BABA-mediated priming for callose was absent in abi4-1 plants (Figure 9), and that exogenous application of ABA mimicked the action of BABA on the level of pathogen resistance and callose accumulation. These results indicate that ABA signalling plays a regulatory role in the primed callose response during BABA-induced resistance. This conclusion is supported by the isolation of a BABA-insensitive mutant carrying a mutation in the 3′ untranslated region of a gene that encodes the ABA biosynthetic enzyme zeaxanthin epoxidase (Toquin et al., 2002). Characterisation of this so-called bai38 mutant revealed that it is impaired in the expression of BABA-induced resistance against H. parasitica and salt stress (Toquin et al., 2002; J. Ton, unpublished results). Because expression of BABA-induced resistance against H. parasitica also correlates with enhanced callose deposition (Zimmerli et al., 2000), the inability of mutant bai38 to express BABA-induced resistance against H. parasitica supports our conclusion that ABA signalling regulates BABA-mediated priming for callose deposition.

Apart from the well-established role of ABA in the regulation of plant responses to abiotic stress (Xiong et al., 2002), there is increasing evidence that ABA regulates responses to pathogen attack. Audenaert et al. (2002) reported that treatment with ABA enhanced the level of susceptibility in tomato to B. cinerea. Additionally, they found that the ABA-deficient tomato mutant sitiens exhibited enhanced resistance against B. cinerea, which correlated with increased activity of phenylalanine ammonia lyase (PAL) during pathogen infection. In addition, these sitiens plants exhibited increased sensitivity to BTH treatment in the transcriptional activation of the PR-1 gene. Apparently, ABA partially represses pathogen-induced activation of SA-dependent defences in tomato. Indications for such negative cross-talk between ABA and SA were also found in Arabidopsis. Mohr and Cahill (2003), who reported that treatment of Arabidopsis with ABA, conferred partial loss of resistance against an avirulent isolate of P. syringae pv. tomato, carrying the avirulence gene avrRpt2. The authors speculated that ABA inhibits RPS2-induced resistance by repressing the SA- and NPR1-dependent defence pathway. In this study, we found that ABA has a positive effect on the level of resistance against P. cucumerina in Arabidopsis. We not only showed that mutations in the ABA signalling pathway impair BABA-induced resistance against P. cucumerina, but also found that treatment with ABA induced resistance against A. brassicicola and P. cucumerina, which coincided with enhanced callose depositions at the sites of pathogen attack (Figure 9). Apparently, ABA represses SA-dependent defence responses, but at the same time, primes for enhanced callose deposition upon pathogen attack. The results obtained in this study show that ABA-dependent priming for callose effectively resists necrotrophic pathogens that are relatively insensitive to SA-inducible resistance. Regarding the recent finding that callose production suppresses SA action (Nishimura et al., 2003), it is tempting to speculate that the negative action by ABA on SA signalling is mediated by enhanced callose accumulation. It remains nevertheless puzzling how BABA can prime for SA-inducible PR-1 expression and ABA-dependent callose deposition at the same time (Figures 4 and 9). On the other hand, we could not detect enhanced levels of ABA-inducible RAB18 transcription in BABA-treated plants (Figure 4). This indicates that the expression of BABA-induced resistance against P. cucumerina is not mediated by major rises in ABA levels. We therefore hypothesise that small, localised fluctuations in ABA levels regulate the BABA-mediated callose deposition. These ABA levels are too small to induce detectable amounts of RAB18 gene expression and do not affect the BABA-mediated priming for SA-inducible defences.

It is evident from the present study that BABA triggers a resistance that is mechanistically different from SA-dependent SAR and JA-/ET-dependent ISR. The observed BABA-mediated priming, leading to earlier and stronger callose formation, also explains how various Arabidopsis mutants impaired in different basal defence mechanisms against necrotrophic fungi are effectively protected by BABA. This illustrates the importance of callose as an early defensive barrier against fungal pathogens. Our results also point to a function of ABA in the regulation of primed callose deposition. How BABA optimises the plant's basal defence response by interfering in the defence signalling network requires further research into the cross-communication between ABA- and SA-dependent signalling pathways.

Experimental procedures

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

Biological material

The Arabidopsis accession Col-0 was obtained from Lehle Seeds (Round Rock, TX, USA), and the Col-0 mutants ein2-1, aba1-5 and abi4-1 were obtained from the Nottingham Arabidopsis Stock Centre. Col-0 mutants npr1-1, pad3-1, coi1-1 and pmr4-1 were provided by X. Dong (Duke University, Durham, NC, USA), Jane Glazebrook (University of Maryland, MD, USA), J. Turner (University of East Anglia, Norwich, UK) and M. Nishimura (Stanford University, CA, USA), respectively. The transgenic Arabidopsis NahG line was obtained from J. Ryals (Novartis Research Triangle Park, USA). All plant genotypes except coi1-1 were germinated on a pasteurised mixture of commercial potting soil and perlite (3 : 1). To select homozygous coi1/coi1 plants, heterozygous COI1/coi1 segregants were germinated on 0.5× Murashige and Skoog medium (Duchefa B.V. Haarlem, the Netherlands) containing 50 µm JA. At 2 weeks after germination, seedlings of all genotypes were individually transferred to 33-ml pots containing the 3 : 1 soil:perlite mixture. Plants were cultivated at 20°C day/18°C night temperatures with 8.5 h of light per 24 h and 60% relative humidity (RH). To prevent wilting of the aba1-5 mutant under these conditions, aba1 plants were maintained at higher humidity by keeping transparent lids on the trays. A. brassicicola strain MUCL 20297, kindly provided by B.P.H.J. Thomma (Katholieke Universiteit Leuven, Belgium), and P. cucumerina, isolated from naturally infected Arabidopsis (accession Landsberg erecta), were grown on 19.5 g l−1 potato dextrose agar (Difco, Detroit) at room temperature for 2 weeks before spores were collected and suspended in 10 mm MgSO4.

Induced resistance bioassays

Treatments with BABA, JA, ABA, salt (NaCl) and BTH were performed with 5–6-week-old plants. Chemicals were dissolved in water and applied as soil drench at the indicated concentrations. Control plants were treated with an equal volume of water. Challenge inoculation was performed 2 days after chemical treatment by applying 6-µl droplets containing 2 × 106 spores ml−1 (A. brassicicola) or 5 × 106 spores ml−1 (P. cucumerina) to six to eight fully expanded leaves. After challenge inoculation, plants were maintained at 100% RH. Disease symptoms were evaluated 4–6 days after inoculation by determining the average lesion diameters in 20–30 plants per treatment. Pathogen colonisation was examined microscopically at 2 days after inoculation in infected leaves that were stained by lactophenol–trypan blue (Koch and Slusarenko, 1990). To determine the effect of the callose inhibitor 2-DDG on BABA-induced resistance against A. brassicicola, leaves of 5-week-old pad3-1 plants were detached and placed in Eppendorf tubes containing 1 ml of the different BABA and 2-DDG solutions. After 24 h incubation, 20 leaves per treatment were challenge-inoculated. At 2 days after challenge inoculation, five leaves were collected for callose staining. Disease symptoms were evaluated in the remaining 15 leaves at 4 days after inoculation.

Quantification of callose deposition

Leaves were collected at the time points indicated and incubated overnight in 95% ethanol. De-stained leaves were washed in 0.07 m phosphate buffer (pH 7), incubated for 15 min in 0.07 m phosphate buffer containing 0.005% calcofluor (fluorescent brightener; Sigma, USA) and 0.01% aniline blue, and subsequently washed in 0.07 m phosphate buffer containing only 0.01% aniline blue (pH 7) to discard excess amounts of the calcofluor. Observations were performed with an epifluorescence microscope with UV filter (BP, 340–380 nm; LP, 425 nm). Callose depositions in response to A. brassicicola infection were quantified by determining the average percentage of callose-inducing spores per infected leaf (n = 8). Callose deposition in response to P. cucumerina infection was quantified from digital photographs of aniline blue-stained leaves (600× final magnification; 150 pixels inch−1). Yellow spots corresponding to stained callose were analysed for number of pixels using adobe photoshop 5.5 software. Callose intensity was expressed as the average number of yellow pixels/million pixels on digital photographs of infected leaf areas (n = 8).

Camalexin quantification

The method for camalexin detection was based on the procedure described by Glazebrook and Ausubel (1994). For each sample, six infected leaves from three different plants were pulverised in liquid N2 and incubated for 25 min in 80% methanol at 55°C. After centrifugation (10 000 g for 5 min), the supernatant was evaporated under vacuum. The residue was extracted two times with 100 µl of chloroform, after which both fractions were combined and evaporated under vacuum. The residue was dissolved in 15 µl of chloroform and spotted on silica gel thin layer chromatography (TLC) plates (Silica gel 60 F254; Merck, Darmstadt, Germany), which were developed with ethylacetate:hexane 9 : 1 (v/v). After drying, camalexin spots were marked under UV light (approximately 345 nm), scraped off and taken up in 2 ml of 100% methanol. Fractions were scanned in a luminescence/fluorescence spectrometer (Perkin Elmer, Buckinghamshire, UK) at an excitation wavelength of 315 nm and an emission wavelength of 385 nm. Camalexin quantities were expressed relative to the sample containing the highest emission value.

RNA gel blot analyis

Total RNA was extracted by homogenising frozen leaf tissue in extraction buffer (0.35 m glycine, 0.048N NaOH, 0.34 m NaCl, 0.04 m EDTA, 4% (w/v) SDS; 1 ml g−1 leaf tissue). The homogenates were extracted with phenol and chloroform and the RNA was precipitated using LiCl, as described by Sambrook et al. (1989). For RNA gel blot analysis, 12.5 µg of RNA was denatured using glyoxal and DMSO (Sambrook et al., 1989). Subsequently, samples were electrophoretically separated on 1.5% agarose gels and blotted onto Hydobond N+ membranes (Amersham, ‘s-Hertogenbosch, the Netherlands) by capillary transfer. The electrophoresis and blotting buffers consisted of 10 and 25 mm sodium phosphate (pH 7.0), respectively. RNA gel blots were hybridised and washed as described previously by Pieterse et al. (1994). DNA probes were labelled with α-32P-dCTP by random primer labelling (Feinberg and Vogelstein, 1983). Probes to detect PDF1.2 and RAB18 transcripts were prepared by PCR with primers based on GenBank sequences At5g44420 and At5g66400, respectively. Probes to detect PR-1 transcripts were derived from a PR-1 cDNA clone (Uknes et al., 1992). Equal loading was visualised by ethidium bromide staining of rRNA.

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

We thank Drs F. Mauch and J.-M. Neuhaus for critical reading of the manuscript, and we are grateful to Sven Daddie, Romain Dubresson and Ronald van Doorn for technical assistance and Dr Gabor Jakab for useful suggestions. We also acknowledge Marc Nishimura and Laurent Zimmerli for providing pmr4-1 seeds and sharing unpublished results. This work was supported by Grant no. 31-064024 from the Swiss National Science Foundation and by the National Center of Competence in Research (NCCR): Plant survival in natural and agronomical ecosystems.

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  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
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