Article15 February 2007free access Pseudomonas syringae pv. tomato hijacks the Arabidopsis abscisic acid signalling pathway to cause disease Marta de Torres-Zabala Marta de Torres-Zabala Biology Division, Imperial College London, Wye Campus, Wye, UK Present address: School of Biosciences, University of Exeter, Stocker Road, Exeter, EX4 4QD UK Search for more papers by this author William Truman William Truman Biology Division, Imperial College London, Wye Campus, Wye, UK Present address: School of Biosciences, University of Exeter, Stocker Road, Exeter, EX4 4QD UK Search for more papers by this author Mark H Bennett Mark H Bennett Biology Division, Imperial College London, Wye Campus, Wye, UK Search for more papers by this author Guillaume Lafforgue Guillaume Lafforgue Biology Division, Imperial College London, Wye Campus, Wye, UK Search for more papers by this author John W Mansfield John W Mansfield Biology Division, Imperial College London, Wye Campus, Wye, UK Search for more papers by this author Pedro Rodriguez Egea Pedro Rodriguez Egea Instituto de Biologia Molecular y Celular de Plantas, Universidad Politecnica—CSIC, Camino de Vera, Valencia, Spain Search for more papers by this author Laszlo Bögre Laszlo Bögre School of Biological Sciences, Royal Holloway, University of London, Egham, Surrey, UK Search for more papers by this author Murray Grant Corresponding Author Murray Grant Biology Division, Imperial College London, Wye Campus, Wye, UK Present address: School of Biosciences, University of Exeter, Stocker Road, Exeter, EX4 4QD UK Search for more papers by this author Marta de Torres-Zabala Marta de Torres-Zabala Biology Division, Imperial College London, Wye Campus, Wye, UK Present address: School of Biosciences, University of Exeter, Stocker Road, Exeter, EX4 4QD UK Search for more papers by this author William Truman William Truman Biology Division, Imperial College London, Wye Campus, Wye, UK Present address: School of Biosciences, University of Exeter, Stocker Road, Exeter, EX4 4QD UK Search for more papers by this author Mark H Bennett Mark H Bennett Biology Division, Imperial College London, Wye Campus, Wye, UK Search for more papers by this author Guillaume Lafforgue Guillaume Lafforgue Biology Division, Imperial College London, Wye Campus, Wye, UK Search for more papers by this author John W Mansfield John W Mansfield Biology Division, Imperial College London, Wye Campus, Wye, UK Search for more papers by this author Pedro Rodriguez Egea Pedro Rodriguez Egea Instituto de Biologia Molecular y Celular de Plantas, Universidad Politecnica—CSIC, Camino de Vera, Valencia, Spain Search for more papers by this author Laszlo Bögre Laszlo Bögre School of Biological Sciences, Royal Holloway, University of London, Egham, Surrey, UK Search for more papers by this author Murray Grant Corresponding Author Murray Grant Biology Division, Imperial College London, Wye Campus, Wye, UK Present address: School of Biosciences, University of Exeter, Stocker Road, Exeter, EX4 4QD UK Search for more papers by this author Author Information Marta de Torres-Zabala1,4, William Truman1,4, Mark H Bennett1, Guillaume Lafforgue1, John W Mansfield1, Pedro Rodriguez Egea2, Laszlo Bögre3 and Murray Grant 1,4 1Biology Division, Imperial College London, Wye Campus, Wye, UK 2Instituto de Biologia Molecular y Celular de Plantas, Universidad Politecnica—CSIC, Camino de Vera, Valencia, Spain 3School of Biological Sciences, Royal Holloway, University of London, Egham, Surrey, UK 4Present address: School of Biosciences, University of Exeter, Stocker Road, Exeter, EX4 4QD UK *Corresponding author. School of Biosciences, University of Exeter, Geoffery Pope Building, Stocker Road, Exeter EX4 4QH, UK. Tel.: +44 1392263789; Fax: +44 1392263434; E-mail: [email protected] The EMBO Journal (2007)26:1434-1443https://doi.org/10.1038/sj.emboj.7601575 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info We have found that a major target for effectors secreted by Pseudomonas syringae is the abscisic acid (ABA) signalling pathway. Microarray data identified a prominent group of effector-induced genes that were associated with ABA biosynthesis and also responses to this plant hormone. Genes upregulated by effector delivery share a 42% overlap with ABA-responsive genes and are also components of networks induced by osmotic stress and drought. Strongly induced were NCED3, encoding a key enzyme of ABA biosynthesis, and the abscisic acid insensitive 1 (ABI1) clade of genes encoding protein phosphatases type 2C (PP2Cs) involved in the regulation of ABA signalling. Modification of PP2C expression resulting in ABA insensitivity or hypersensitivity led to restriction or enhanced multiplication of bacteria, respectively. Levels of ABA increased rapidly during bacterial colonisation. Exogenous ABA application enhanced susceptibility, whereas colonisation was reduced in an ABA biosynthetic mutant. Expression of the bacterial effector AvrPtoB in planta modified host ABA signalling. Our data suggest that a major virulence strategy is effector-mediated manipulation of plant hormone homeostasis, which leads to the suppression of defence responses. Introduction Plant–pathogen interactions involve a dynamic process of cut, thrust and counterthrust as the host attempts to contain the insurgent within the complex intracellular and extracellular landscape of the battlefield. Plant basal defences provide several strategic layers of constitutive and induced defences, the latter based upon the ability to detect and respond to conserved molecular patterns associated with the invading microbe. The various repertoires of predominately surface-expressed ligands, commonly referred to as PAMPs (pathogen-associated molecular patterns), engage different combinations of plant-surface receptors whose output is customised to reflect the degree of ‘danger’ posed by the particular pathogen. The basal defense response manifests itself in the form of biochemical and structural defences designed to restrict microbial multiplication and nutrition, including rapid ionic changes and phosphorylation cascades, ultimately leading to transcription of defence-related genes and the formation of defensive cell wall depositions, termed papillae (Nurnberger et al, 2004; Zipfel and Felix, 2005). The frontline weapons of bacterial virulence comprise a collection of chemical virulence factors and ∼40 proteinaceous ‘effectors’. The latter are delivered into the plant cell via a type III protein secretion system (T3SS; Mudgett, 2005). Evidence is emerging that the type III effectors (T3Es) interfere with host signalling and metabolism to promote suppression of basal defence and pathogen nutrition. However, specific function has been assigned to very few T3Es, and our knowledge of the synergistic collaborations with other effectors that may be required for successful parasitism is rudimentary. Three phytohormones, salicylic acid (SA), jasmonic acid (JA) and ethylene (ET), are known to participate in regulating defence responses in plants. SA is predominately associated with resistance against biotrophic and hemi-biotrophic pathogens and the establishment of systemic acquired resistance (Grant and Lamb, 2006). By contrast, JA- and ET-dependent defence mechanisms generally contribute to resistance against necrotrophic pathogens, suggesting that the signalling network engaged by the host is dependent upon the nature of the pathogen and its mode of pathogenicity (Glazebrook, 2005). The plant hormone abscisic acid (ABA) is involved in plant responses to several abiotic stresses (drought, salt and cold) as well as seed germination and plant growth (Seo and Koshiba, 2002; Nambara and Marion-Poll, 2005). Additionally, exogeneous ABA treatment increases the susceptibility of various plant species to bacterial and fungal pathogens (Henfling et al, 1980; Ward et al, 1989; McDonald and Cahill, 1999; Mohr and Cahill, 2003; Thaler and Bostock, 2004). ABA-deficient mutants showed a reduction in susceptibility to the necrotroph Botrytis cinerea (Audenaert et al, 2002) and virulent isolates of Pseudomonas syringae pv tomato DC3000 in tomato (Thaler and Bostock, 2004), and the oomycete Hyaloperonospora parasitica in Arabidopsis (Mohr and Cahill, 2003). Collectively, these data suggest that ABA behaves as a negative regulator of defence responses. We are interested in how virulence factors (including T3Es) produced by DC3000 in Arabidopsis suppress plant basal defences. Building upon analysis of genome-wide transcriptome changes during early stages of the A. thaliana/DC3000 interaction (Truman et al, 2006) we now present results supporting effector-mediated manipulation of ABA biosynthetic and signalling pathways as a core virulence mechanism. The changes in plant hormones observed during infection by bacteria and fungi have traditionally been considered as ‘side effects of successful parasitism’. By contrast, we now show that the successful manipulation of the ABA hormonal network has probably been a fundamental step in the evolution of a plant pathogenic bacterium. Results Microarrays reveal a role for ABA in virulence We have analysed previously the temporal dynamics of host gene expression during bacterial infection of Arabidopsis leaves by global transcriptional profiling (Truman et al, 2006). Three challenges designed to report background inoculation effects (mock; 10 mM MgCl2), PAMP responses (DC3000hrpA−, a mutant compromised in T3SS) or disease (wild-type DC3000), and carefully chosen sampling times enabled the identification of differentially expressed genes associated with the activation of basal defence and establishment of pathogenesis. By removing the basal defence signature attributable to challenge with the hrpA− mutant, we captured the specific effects of bacterial virulence factors on host transcription. Hierarchical clustering identified transcripts coregulated by T3E delivery 12 h post-inoculation (12 hpi). These transcripts represent genes upregulated by T3Es, either due to the action of the effectors themselves or, alternatively, as a host response to T3E activities. We have now examined in detail one coregulated cluster consisting of 47 T3E-induced genes (Table I). The group of genes is characterised by an over-representation of transcripts encoding protein phosphatases of the 2C class (PP2C), which are implicated in responses to ABA, including ABI1 (abscisic acid insensitive 1; Schweighofer et al, 2004). Remarkably, NCED3 (At3g14440), which encodes the enzyme that catalyses the early limiting step in water stress-induced ABA biosynthesis (Qin and Zeevaart, 1999; Tan et al, 2003), was induced more than 15-fold by T3Es. Other ABA response-associated genes resident in this cluster include AFP (abscisic acid insensitive-5 binding protein), an ABA signalling regulator (At1g69260) (Lopez-Molina et al, 2003), and four NAC (for NAM, ATAF1, 2 and CUC2) transcription factors (At1g52890, At3g15500, At4g27410 [RD26] and At5g39610) previously shown to be ABA, drought and NaCl-inducible (Tran et al, 2004). Table 1. A coregulated set of T3E-induced genes responsive to ABA identified by hierarchial clustering AGI number Function (name) ABA/stress reference ARE FC ABA biosynthetic genes At3g14440 ABA synthesis (NCED3) Qin and Zeevaart (1999) B 17.0 Protein phosphatase 2C clade A At5g59220 PP2C Xin et al (2005) 2D 10.0 At1g72770 PP2C (HAB1) Saez et al (2004) 2A2D 2.1 At1g07430 PP2C AB4D 8.7 At3g11410 PP2C (AtPP2CA) Kuhn et al (2006) AC2D 4.2 At5g57050 PP2C (ABI2) Merlot et al (2001) B 3.2 At4g26080 PP2C (ABI1) Merlot et al (2001) C 2.9 NAC domain transcription factors At5g39610 NAC TF (AtNAC6) He et al (2005) 3.6 At1g52890 NAC TF (ANACO19) Tran et al (2004) 3C2D 2.9 At3g15500 NAC TF (AtNAC3) Tran et al (2004) C3D 7.9 At4g27410 NAC TF (RD26) Fujita et al (2004) A2CD 4.5 Transcription factors of various families At1g66550 WRKY TF (WRKY67) D 10.2 At5g49450 bZIP family TF 2D 4.3 At1g24440 C3HC4 zinc finger? 3.1 Other signalling pathways At1g69260 ABI5 binding protein Lopez et al (2003) 2C2D 3.1 At1g05100 MAP3K (MAPKKK18) BC3D 10.9 At2g02710 PAC motif containing BD 2.1 At5g62540 Ubiquitin conjugating D 2.2 Assorted physiological processes At2g03760 Steroid sulphotransferase 7.0 At5g13750 Transporter (ZIFL1) C2D 2.7 At3g11340 UDP glucosyl transferase 2.4 At3g46660 UDP glucosyl transferase D 5.3 At3g06500 β-Fructofuranosidase B3D 4.3 At4g21680 Oligopeptide transporter 2.5 At1g05560 UDP-glucose transferase Tran et al (2004) D 3.3 At1g26770 Expansin (AtEXPA10) 4.4 At5g13820 Telomeric DNA-binding-1 Nagaoka and Takano (2003) C3D 3.2 At3g14660 Cytochrome P450 C 2.1 Genes of unknown function At3g01650 Copine related 3.3 At1g24600 Expressed protein A2D 3.2 At3g48350 Cysteine proteinase like 3D 8.1 At3g28007 Nodulin MtN3 family 4D 2.7 At3g29575 Expressed protein 4C3D 3.3 At1g69480 ERD1/XPR1/SYG1 BC 3.4 At2g28400 Expressed protein 3.2 At4g33980 Expressed protein 3C 2.4 At5g04250 OTU cysteine protease? C3D 5.1 At5g54730 WD40 repeat protein 3D 3.9 At5g42900 Expressed protein C2D 11.5 At1g33110 MATE efflux protein 3.1 At5g64230 Expressed protein D 6.2 At5g65660 HPRG protein 2D 2.8 At1g58270 MATH domain protein 2A4D 3.0 At3g07350 Expressed protein ACD 6.5 At3g28210 Zinc-finger protein Xin et al (2005) D 2.1 At5g13360 Auxin responsive GH3 D 2.1 At5g64250 2-Nitropropane diox. 2.7 Each of the 880 genes, significantly differentially expressed in response to T3E at 12 hpi, were median-centred and logged before clustering. This table presents a cluster of 47 genes containing a significant over-representation of ABA response elements in their promoters (1 kb upstream). Most genes of known function had previously been associated with abiotic stress. ARE—presence or absence of an ABA response element as defined in the PLACE database (Higo et al, 1999). Scoring is non-redundant and the number of occurrences precedes the motif. (A) ABREMOTIFAOSOSEM—TACGTGTC; (B) ABREATCONSENSUS—[CT]ACGTGGC; (C) ACGTABREMOTIFA2OSEM—ACGTG[GT]C; (D) ABRELATEDD1—ACGTG. FC—fold change. Analysis of the proximal 1 kb promoter regions of the coregulated genes using the PLACE Signal Scan programme (Higo et al, 1999) identified that more than 50% contain one or more ABA response elements (ABRE; ACGTG[GT]C), which could be further elaborated into three overlapping regions of increasing complexity. All three contained the core ACGTG motif, which itself was present in more than 75% of the promoters (Table I). The significant over-representation of these motifs was confirmed using POBO (Kankainen and Holm, 2004). In summary, our analysis suggested that one mechanism of T3E action is to elevate components of the ABA signalling and biosynthesis pathways. ABA levels in challenged tissues In a compatible interaction leading to disease, the induction of NCED3 between 4 and 12 hpi follows T3E delivery. As NCED is a key regulatory enzyme in ABA biosynthesis, we examined whether T3Es were directly manipulating levels of ABA. We measured ABA at 8, 12 and 18 h following mock, DC3000 or DC3000 hrpA− challenges. Increases in ABA occurred rapidly, and by 18 hpi ABA levels were ∼10-fold higher in DC3000 than in hrpA− or mock-challenged leaves (Figure 1A) and were directly correlated to the level of DC3000 inoculum (Figure 1B). Although DC3000 multiplies in planta, there was no significant difference in bacterial numbers (hrpA− versus DC3000) at 10 hpi despite increasing ABA levels (data not shown), and 15-fold less virulent bacteria produced more than six-fold more ABA than the non-pathogenic hrpA− mutant (Figure 1B). As the hormones SA and JA have already been demonstrated to play a role in plant defences, we compared SA, JA and ABA levels 18 hpi in uninoculated and pathogen-challenged leaves (Figure 1C). Foliar ABA and JA were significantly induced by DC3000, suggesting DC3000 induces both ABA and JA biosynthesis, whereas free SA did not differ significantly between hrpA− and DC3000. Figure 1.ABA levels increase during a compatible interaction in an inoculum concentration-dependent manner. (A) Time course of changes in ABA levels in Col-5 leaves after mock infiltration (MgCl2, 10 mM) or challenge with DC3000 or DC3000 hrpA−. (B) ABA levels are dependent on DC3000 concentration. ABA levels in leaves challenged with DC3000 hrpA− or increasing concentrations of DC3000 were measured 18 hpi. (C) Relative in planta levels of ABA, JA and SA during compatible (DC3000) and basal defence (DC3000hrpA−) responses. Hormones were measured in both non-inoculated and challenged (0.5 × 108 CFU/ml) Col-5 leaves at 18 hpi. (D) 9-cis-Epoxycarotenoid-dioxygenase3 (NCED3) is induced by T3Es concomitant with elevated foliar ABA. Time course of NCED3 steady-state mRNA levels following DC3000 and DC3000hrpA− challenge determined, in independent experiments, by RNA blot (upper panel) or RT–PCR. Bacterial titres and RT–PCR values (copies of target transcript/copy actin 2) are means of triplicate samples and error bars represent 1 s.d. All experiments were repeated at least twice with similar results. Download figure Download PowerPoint NCED3, strongly induced by T3Es at 12 hpi, encodes the major stress-induced ABA regulatory enzyme, and is the most likely candidate to contribute to elevated ABA levels in the compatible interaction. In agreement with our microarray results NCED3, steady-state levels increased from 8 hpi only after challenge with DC3000 (Figure 1D). The transient increase in NCED3 transcript at 2 hpi evident in both DC3000 and DC3000hrpA−-challenged leaves resulted from the inoculation procedure, as it was also induced by mock infiltration (data not shown). ABA application and pathogenicity responses share significant overlap To identify common ABA signalling components induced by T3Es we interrogated experiments in the NASC arrays (http://affymetrix.arabidopsis.info) database indicative of host responses to hormone application or abiotic and biotic stresses. Hierarchical clustering of the 880 genes significantly differentially regulated between DC3000 and DC3000hrpA− challenges at 12 hpi closely associated abiotic stress experiments with compatibility. In addition to ABA treatments at 30 min, 1 and 3 h, genes modified by T3Es were also found to respond to salt and osmotic stress treatments (Figure 2A). A major inducible cluster (i) was shared between compatible responses (DC3000 challenge at 6, 12 and 24 hpi) and osmotic stress. This cluster contains a subcluster (ii) that comprises the majority of the genes represented in Table I, including seven PP2C-s, NCED3 and three NAM-s (annotated in Supplementary Table I). Two other notable clusters represent genes commonly suppressed both by T3Es and osmotic stress (iii) and genes differing in response between osmotic stress and T3Es activity (iv). Figure 2.Hierarchal clustering of T3E-responsive transcripts shows a strong overlap with abiotic stresses. (A) The 880 probesets identified previously as significantly differentially expressed in response to T3Es at 12 hpi were clustered using GeneChip expression data from the AtGenExpress consortium. Experiments reporting the effects of cold, drought, salt stress, osmotic stress (mannitol) and ABA application were included as well as additional time points reporting T3E activity. All data sets were normalised and interpreted using the GCRMA function of the Bioconductor microarray analysis package (http://www.bioconductor.org/). Hierarchical clustering was applied using an uncentred correlation and complete linkage clustering. Genes induced relative to their control are coloured red, those suppressed are coloured green, whereas genes unchanged in their expression levels are coloured black. Cluster i, genes sharing strong similarity to regulatory networks induced by abiotic stresses, in particular, osmotic stress. Cluster ii, all the Clade A PP2Cs originally identified in Table I are highlighted in blue. Cluster iii, genes suppressed by both T3E and abiotic stresses. Cluster iv, genes suppressed by T3E but induced by abiotic stresses. (B) Venn diagram showing the commonality between transcripts differentially induced by T3Es and those strongly induced by ABA or MeJA application. SAM (Tusher et al, 2001) was used to identify genes induced by DC3000 relative to DC3000hrpA− or DC3000hrcC at 6, 12 and 24 hpi from two independent but similar experiments, with a minimum fold change of 2 and a false discovery rate of less than 5%. ABA- and MeJA-induced genes were identified solely on fold change taking the average of two replicates. Download figure Download PowerPoint Given that T3Es increase foliar ABA and JA levels by 18 hpi (Figure 1) and the strong similarity between transcripts induced by ABA and T3Es (Figure 2A), we examined the overlap between responses to each hormone treatment and T3Es using experiments from the NASC database repository. The ABA response has a substantial overlap with T3E-upregulated transcripts (42%). Interestingly, 41% of the transcripts responsive to JA are also T3E induced (Figure 2B, Supplementary Table II). Collectively, these data suggest transcriptional reprogramming induced by T3E activity is highly similar to that induced through the ABA response pathway, and that ABA as well as JA signalling pathways contribute to pathogenicity. Exogenous application of ABA enhances multiplication of wild-type DC3000 and a hrpA− mutant The effect of exogenous application of ABA on the multiplication of DC3000 and DC3000hrpA− in challenged leaves was examined. If de novo ABA biosynthesis contributed to DC3000 virulence, we hypothesised that ABA application would allow the hrpA− mutant to overcome host basal defences. ABA pretreatment 24 h before bacterial inoculation increased DC3000 virulence by an order of magnitude (Figure 3C). Furthermore, ABA application also enabled hrpA− bacteria partially to overcome basal defences, consistently permitting a ∼five-fold increase in growth (Figure 3A, statistical significance confirmed by ‘t’-tests). Thus, exogenously applied ABA promotes colonisation by both virulent and normally non-pathogenic P. syringae. Figure 3.Effect of exogenous application of ABA and ABA signalling mutants on the growth of compatible bacteria. (A) Growth of DC3000 and DC3000 hrpA− on Col-0 plants sprayed 24 h before inoculation with ABA (100 μM in 0.2% ethanol) or mock (0.2% ethanol). (B) Growth of DC3000 in the leaves of wild-type Ler and the ABA hypersensitive mutants abi1-sup7 and abi1-sup5. (C) Growth of DC3000 in wild-type Ler and the ABA-insensitive mutants abi1-1, abi2-1 or transgenic 35S∷HAB1 leaves. (D) Growth of DC3000 and DC3000 hrpA− in leaves of wild-type Col-0 and the ABA biosynthetic mutant aao3. Figures are representative of at least three replicate experiments. Error bars represent 1 s.d. Download figure Download PowerPoint ABA signalling mutants in defence To examine the potential role of ABA signalling in pathogenicity, we tested the T3E-induced Clade A PP2Cs ABI1, ABI2 and HAB1 to determine their role in mediating the suppression of basal defense. The dominant mutations abi1-1, abi2-1 as well as 35S∷HAB1 plants show reduced sensitivity to ABA compared with wild type (Kornneef et al, 1984; Saez et al, 2004). Conversely, intragenic revertants of the originally dominant abi1-1 mutant, abi1-1sup5 and abi1-1sup7 behave as recessive alleles of ABI1 (Gosti et al, 1999). These mutants were tested for their ability to support bacterial multiplication. Figure 3B shows that the ABA-hypersensitive lines, abi1-1sup5 and abi1-1sup7, supported up to 30-fold more DC3000 multiplication. Conversely, the ABA-insensitive abi1-1 and abi2-1 mutants and the 35S∷HAB1 transgenic line were 20–80-fold more resistant than wild type (Figure 3C). In contrast to ABA application, no significant growth differences were obtained for DC3000hrpA− (not shown). Arabidopsis ABA aldehyde oxidase 3 (At2g27150; AAO3), a cytosolically localised enzyme, catalyses the last step of ABA biosynthesis in response to drought stress (Seo and Koshiba, 2002). Multiplication of virulent DC3000 in an AAO3 T-DNA knockout line (SALK_072361) was significantly restricted compared with wild type. We interpret these data to suggest that both de novo ABA biosynthesis induced by T3E delivery and PP2C activities collaborate to regulate pathogenicity. ABA attenuates callose deposition associated with basal defence The susceptible phenotype of the ABA hypersensitive abi1suppressor mutants correlated with enhanced chlorosis and formation of water-soaked lesions on leaves 3–4 dpi (days post-infection) (Figure 4A). A hallmark of basal defence to attempted bacterial and fungal penetration is the deposition of callose in paramural deposits. We used aniline blue staining (Keshavarzi et al, 2004) to monitor callose deposition at the cellular level (Figure 4B). Compared with wild-type leaves challenged with DC3000 at 12 hpi (Figure 4Ba), both abi1-sup5 (data not shown) and abi1-sup7 (Figure 4Bb) were completely devoid of callose-associated fluorescence. By contrast, the ABA-insensitive mutants abi1-1 (Figure 4Bc), abi2-1 and the 35S∷HAB1 line (Figure 4Bd) showed augmented callose deposition, although no significant differences in the response were detected in mutant leaves following challenge with DC3000hrpA− (data not shown). Next, mock- (Figure 4Be) and ABA-treated (Figure 4Bf) leaves were compared following virulent DC3000 challenge. As predicted, less callose-associated fluorescence was detected in ABA-treated plants 12 hpi after DC3000 challenge than in control inoculated leaves (compare Figure 4Be with 4Bf). Figure 4.ABA signalling mutants have altered macroscopic and microscopic disease symptoms. (A) ABA-hypersensitive plants display enhanced chlorosis and necrosis 3 dpi following challenge with DC3000 (2.5 × 105 CFU/ml) compared with wild-type Ler. (B) Callose deposition in wild-type Ler (a) contrasts to reduced callose deposition in abi1-sup7 (b) and enhanced callose deposition in abi1-1 mutant and the transgenic 35S∷HAB1 line (c and d, respectively), 12 hpi with DC3000. Callose deposition in mock-treated (e) or ABA-treated (f) wild-type Col-0, 12 hpi with DC3000. Download figure Download PowerPoint ABA suppression of genes involved in basal defense We have shown that effectors delivered intracellularly are able to downregulate certain PAMP-induced genes (PIGs; de Torres et al, 2006; Truman et al, 2006). To examine whether ABA itself contributes to suppression of these basal defence components, we used RNA blot and RT–PCR to examine the expression level of two PIGs suppressed by T3Es, FRK1 (flagellin-induced receptor kinase 1) and the glycerol kinase encoding NHO1 (Kang et al, 2003; Truman et al, 2006) in ABA-insensitive mutants challenged with DC3000 or DC3000hrpA−. Figure 5 shows that suppression of both FRK1 (Figure 5A and B) and NHO1 (Figure 5A and C) is delayed in both abi1-1 and abi2-1 (ABA-insensitive) backgrounds. The effects of the abi1-1 and abi1-2 mutations appear to be to stabilise the relative amounts of defence transcripts. We are therefore unable to determine whether the delayed suppression of defence transcripts is due to enhanced innate immunity or an inability to activate suppression mechanisms. Whichever mechanism is operative, the elevated basal defence transcripts in the compatible interaction are consistent with the enhanced resistance to virulent DC3000 as shown in Figure 2. Figure 5.Suppression of defence gene transcripts is attenuated in ABA-insensitive mutants. (A) RNA blot of FRK1 and NHO1 transcript accumulation over time following DC3000hrpA− or DC3000 challenges of wild-type Ler (left hand panel) or the ABA-insensitive mutant, abi1-1. (B) Independent RT–PCR experiment showing suppression of FRK1 accumulation in the abi1-1 mutant is restricted after challenge with DC3000 compared with hrpA−. (C) RT–PCR reveals that suppression of NHO1 accumulation by DC3000 is also compromised in the abi2-1 mutant. RT–PCR experiments measured copies of target transcript/copy actin 2, and were in triplicate. Error bars represent 1 s.d. Download figure Download PowerPoint Expression of the bacterial effector AvrPtoB in planta increases ABA levels Conditional expression of the conserved P. syringae effector AvrPtoB increases susceptibility to DC3000hrpA−, suppresses callose deposition and dramatically suppresses PIG transcripts (de Torres et al, 2006). Using transgenic plants carrying avrPtoB under the control of a dexamethasone (Dex)-activated promoter, we examined NCED3, NHO1 and FRK1 transcripts in response to MgCl2 or DC3000hrpA− inoculations following Dex induction by RNA blot (Figure 6A) or RT–PCR (Figure 6B). As expected, Dex-treated leaves suppressed induction of NHO1 and FRK1 by DC3000hrpA−. However, avrPtoB expression induced NCED3 irrespective of the inoculation, suggesting that AvrPtoB alone can modify ABA signalling responses (Figure 6A and B). Notably, the levels of NCED3 mRNA induced by Dex are ∼5–6 times the levels obtained in a compatible interaction. Figure 6.The bacteria effector, AvrPtoB, modifies basal defence transcripts and triggers elevated foliar ABA levels. (A, B) Six hours after Dex or mock treatment, Dex∷avrPtoB transgenic plants wer