127 Noble Research Center, Department of Entomology and Plant Pathology, Oklahoma State University, Stillwater, OK 74078, USA
Correspondence
Carol L. Bender
cbender{at}okstate.edu
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ABSTRACT |
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Present address: Pacific Basin Tropical Plant Genetic Resource Management Unit, USDA-ARS, PO Box 4487, Hilo, HI 96720, USA.
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INTRODUCTION |
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Both P. syringae and Pseudomonas aeruginosa produce the exopolysaccharide alginate, a copolymer of O-acetylated -1,4 linked D-mannuronic acid and its C-5 epimer, L-guluronic acid. P. aeruginosa is an important human pathogen that causes opportunistic pulmonary infections in patients suffering from cystic fibrosis. Some strains of P. aeruginosa also cause disease in plants (Rahme et al., 1995
), although the symptoms (soft rotting) and mechanistic aspects of entry into plant tissue are clearly different for P. aeruginosa and P. syringae (Plotnikova et al., 2000
). A critical aspect of pulmonary infection by P. aeruginosa is the conversion of this bacterium to a mucoid, alginate-overproducing phenotype during the chronic-infection stage of cystic fibrosis (Lyczak et al., 2002
). In cystic fibrosis patients, the alginate capsule has been reported to inhibit opsonic and non-opsonic phagocytosis and to enhance bacterial survival during the macrophage-mediated oxidative burst (Cabral et al., 1987
; Simpson et al., 1988
, 1989
).
Studies have shown that alginate functions in the virulence of some P. syringae strains (Osman et al., 1986; Gross & Rudolph, 1987
; Yu et al., 1999
). Alginate production by P. syringae has been associated with increased epiphytic fitness, resistance to desiccation and toxic molecules, and the induction of water-soaked lesions on infected leaves (Fett & Dunn, 1989
; Rudolph et al., 1994
; Yu et al., 1999
). In plants inoculated with the alginate-defective mutant P. syringae pv. syringae 3525.L, symptoms were less severe and bacterial multiplication was reduced relative to the wild-type (Yu et al., 1999
), suggesting that alginate facilitates the colonization and/or dissemination of this strain in planta. Strains of P. syringae, like P. aeruginosa, are normally non-mucoid; however, some of the signals for conversion to the mucoid phenotype differ in these species. For example, copper-based sprays are frequently applied to plants for the control of bacterial diseases and exposure to copper ions dramatically increases alginate production in certain strains of P. syringae but not in P. aeruginosa (Kidambi et al., 1995
). As in P. aeruginosa, increased levels of NaCl and sorbitol activate the transcription of alginate promoters in P. syringae, indicating that elevated osmolarity is a signal for alginate production in both pseudomonads (Keith & Bender, 1999
; Peñaloza-Vázquez et al., 1997
). Another conserved signal for alginate gene expression in P. aeruginosa and P. syringae is exposure to reactive oxygen species (ROS) (Keith & Bender, 1999
; Mathee et al., 1999
).
Although alginate was shown to be the major exopolysaccharide produced by P. syringae in water-soaked lesions (Fett & Dunn, 1989), until recently there was no evidence that alginate gene expression was induced in planta. Boch et al. (2002)
used in vivo expression technology (IVET) to show that a variety of genes are specifically induced during the infection of Arabidopsis thaliana by P. syringae pv. tomato DC3000. One of the plant-inducible genes identified in the IVET screen was algA, which encodes a bifunctional enzyme in the alginate biosynthetic pathway (Shinabarger et al., 1991
). Although the IVET study clearly demonstrated that alginate genes are expressed in A. thaliana, the temporal expression of the algA : : uidA promoter fusion was not investigated (Boch et al., 2002
). The primary objective of our study was to construct a defined algD : : uidA transcriptional fusion and use this to monitor alginate gene expression in host and non-host plants infected with P. syringae pv. tomato DC3000, which is a also a pathogen of tomato and Brassica spp. (e.g. cauliflower, collard) (Moore et al., 1989
; Zhao et al., 2000
). algD encodes GDP-mannose dehydrogenase, which is the committed step in alginate biosynthesis and the first gene to be transcribed in the alginate structural gene cluster of P. syringae (Peñaloza-Vázquez et al., 1997
). In the current study, algD expression was monitored in susceptible hosts (tomato and collard), a resistant host (tomato carrying the PtoR resistance gene) and a non-host plant (tobacco), which undergoes the HR in response to infection with P. syringae pv. tomato.
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METHODS |
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Construction of an algD : : uidA transcriptional fusion.
A genomic library of P. syringae pv. tomato DC3000 was previously constructed in pRK7813 (Boch et al., 2002). In this study, a 3·9 kb KpnI fragment from pSKK3.9, which contains algD from P. syringae pv. syringae FF5 (Peñaloza-Vázquez et al., 1997
), was used to screen the DC3000 genomic library for clones containing algD. A cosmid clone designated pRCK1 contained an 8 kb BamHI fragment that hybridized with the probe. This fragment was subcloned into pBluescript SK(+), resulting in pBRCK1, which was shown by sequence analysis to contain the algD gene and the predicted promoter region (Keith, 2002
). The algD promoter in the related pathogen, P. syringae pv. syringae FF5, was previously defined by deletion analysis (Peñaloza-Vázquez et al., 1997
). The corresponding region in P. syringae pv. tomato DC3000 was sequenced in pBRCK1 and showed 81·6 % nucleotide identity with the algD promoter of FF5 (Keith, 2002
). Therefore, the strategy used for subcloning the DC3000 algD promoter was based on prior results with FF5, since these two strains were highly homologous in their algD upstream regions.
pBBR.Gus, which contains a promoterless glucuronidase (GUS) gene (uidA) downstream of the polylinker in pBBR1MCS, was used to create an algD : : uidA transcriptional fusion. To obtain the algD promoter region in the transcriptionally active orientation, a 1 kb PCR product was cloned into the HindIII/PstI sites of pBBR.Gus. The promoter region was amplified from pBRCK1 by using the forward primer 5'-CGGAAAGCTTTAAACCAGTTCGATG-3' (the HindIII site is underlined) and the reverse primer 5'-CCGCCTGCAGGGTAACAACTAGTTCAG-3' (PstI site is underlined). The amplified region contained 956 nt upstream of the algD translational start site, including the recognition sequence for 22, an alternate sigma factor that mediates transcription at the algD promoter (Keith & Bender, 1999
; Wozniak & Ohman, 1994
). After amplification of the 1 kb PCR product, cloning into pBBR.Gus as a HindIII/PstI fragment, and transformation into E. coli DH5
, plasmid pDCalgDP was recovered. Expression of the algD promoter was also monitored in P. syringae pv. tomato DC3000-hrcC, a Cmr strain. In these experiments, the algD promoter was subcloned from pDCalgDP as a 1 kb HindIII/PstI fragment into pBBR.Gus.Km (Table 1
), resulting in pDCalgDP.Km. The presence of the algD promoter region in both pDCalgDP and pDCalgDP.Km was confirmed by sequence analysis.
Quantitative GUS assays.
Liquid cultures of DC3000(pBBR.Gus) and DC3000(pDCalgDP) were incubated at 28 °C in MG broth supplemented with 25 µg chloramphenicol ml-1. Each strain carrying an individual construct was inoculated into triplicate aliquots of medium (100 ml MG broth) and incubated at 28 °C. At different time points (0, 12, 24 and 48 h), a 1 ml sample of bacterial cells was removed from each tube and assayed for GUS activity as described previously (Peñaloza-Vázquez & Bender, 1998). The protein concentration of cell lysates was determined using the Bio-Rad protein assay kit, following the manufacturer's protocol. GUS activity was expressed in units (U) (mg protein)-1, with 1 U equivalent to 1 nmol of methylumbelliferone formed min-1.
Plant growth and inoculation procedures.
Tomato (Lycopersicum esculentum cv. Rio Grande-PtoS and -PtoR) and collard (Brassica oleracea var. viridis L. cv. Vates) seedlings were maintained in a growth chamber at 2425 °C in 3040 % relative humidity, with a photoperiod of 12 h. Plants were maintained at 90 % relative humidity for 48 h before inoculation. Derivatives of P. syringae pv. tomato DC3000 were grown at 28 °C for 48 h on MG agar supplemented with the appropriate antibiotics and cells were resuspended to an OD600 of 0·3 (
5x108 c.f.u. ml-1) in sterile distilled water. Silwet L77 (Osi Specialties) was added to the bacterial inoculum at a concentration of 0·2 µg ml-1. Six-week-old plants were spray-inoculated with an airbrush (55·2 kPa) until leaf surfaces were uniformly wet. After inoculation, tomato and collard plants were incubated at 24 °C in 60 % relative humidity with a 12 h photoperiod for the duration of the experiment.
Tobacco (Nicotiana tabacum cv. Petite Havana) leaves were infiltrated (OD600=0·1) with selected bacterial strains using established methods (Schaad, 1988).
Histochemical detection of GUS activity.
Tomato and collard leaves were sampled at 0, 24, 48, 72, 120 and 168 h post-inoculation (p.i.) and vacuum-infiltrated with a substrate surfactant solution [5-bromo-4-chloro-3-indoyl -D-glucuronide (X-gluc), 0·5 mg ml-1, and L77 at 0·2 µl ml-1 in 50 mM sodium phosphate buffer, pH 7·0]. Vacuum-infiltrated leaves were incubated at 37 °C overnight and fixed and destained in 80 % ethanol at 37 °C (Hugouvieux et al., 1988). Samples from infiltrated tobacco leaves were excised at 2, 4, 6, 8, 10 and 12 h p.i. with a sterile cork borer (1 cm diam.) and assayed for GUS activity as described above.
Determination of bacterial growth in planta.
In experiments designed to follow the total population of the DC3000 strains in collard and tomato, random leaf samples were taken at the time points indicated above. Each leaf was weighed separately (three replicates per time point) and macerated in 2 ml (tomato) or 5 ml (collard) sterile distilled H2O. Bacterial populations in tobacco leaves were monitored by removing leaf disks (1 cm diameter) at 0, 6 and 12 h after infiltration with DC3000 strains. Bacterial counts were determined by plating dilutions of the leaf homogenate onto MG agar supplemented with 25 µg chloramphenicol ml-1. Colonies were counted after incubating the plates for 4896 h and the experiment was then repeated twice. Bacterial population counts were similar on media with and without antibiotic selection, indicating that the vector was stably maintained in planta.
Detection of .
Tobacco leaves were monitored for the superoxide anion () by infiltration with nitro blue tetrazolium (NBT) using methods similar to those described by Doke (1983)
. Tobacco leaf disks were excised at 2, 4, 6, 8, 10 and 12 h after inoculation with DC3000, DC3000-hrcC or sterile distilled H2O. Leaf disks were then vacuum-infiltrated with 0·05 M sodium phosphate buffer (pH 7·5) containing 0·5 mM NBT. Leaf tissue was incubated at ambient temperature under cool fluorescent lighting for 20 min and the reaction was then stopped with 95 % ethanol. Chlorophyll was removed by repeated changes of 95 % ethanol.
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RESULTS |
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Symptoms and algD expression in collard leaves
Collard leaves inoculated with P. syringae pv. tomato were examined for disease symptoms and GUS activity to determine whether the manifestation of a particular symptom was correlated with alginate gene expression. In collard leaves inoculated with DC3000(pBBR.Gus) and DC3000(pDCalgDP), water-soaked lesions were first visible at 72 h p.i. (Fig. 1i), and chlorosis and necrosis were evident beginning at 7296 h p.i. (Fig. 1b
). Histochemical staining was used to study expression of the algD promoter in planta. When the algD promoter is activated, GUS is produced and leaves incubated with the chromogenic substrate X-gluc stain blue where the bacteria are located. At 72 h p.i., the water-soaked lesions on collard leaves inoculated with DC3000(pDCalgDP) (Fig. 1i
) stained blue when the tissue was infiltrated with X-gluc (Fig. 1j
). Collard leaves inoculated with DC3000(pBBR.Gus) did not stain when treated with X-gluc, regardless of the time following inoculation (Fig. 1l
). Both DC3000(pBBR.Gus) and DC3000(pDCalgDP) grew equally well in collard reaching a population of 5x109 c.f.u. g-1 in 120 h (Fig. 1m
), which indicates that the constructs pBBR.Gus and pDCalgDP did not have a negative impact on the growth of DC3000 in planta.
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When tobacco cv. Petite Havana was infiltrated with DC3000(pDCalgDP) (Fig. 4a) and DC3000(pBBR.Gus) (not shown), an HR was visualized within 12 h p.i. An HR was also visualized in tobacco leaves inoculated with DC3000 containing pCPP2438, which contains the hrpA promoter fused to a promoterless GUS gene (Fig. 4a
). The hrpA gene encodes the major subunit of the TTSS appendage known as the Hrp pilus and is essential for the physical translocation of virulence effectors (Jin & He, 2001
; Wei et al., 2000
). After vacuum infiltration with X-gluc, incubation and destaining, a high level of GUS activity was observed in tobacco tissue inoculated with DC3000(pCPP2438) at 212 h p.i. (Fig. 4c
). Activation of the algD : : uidA fusion was delayed in comparison to the hrpA : : uidA fusion, with GUS activity first appearing 8 h p.i. (Fig. 4c
). No GUS expression was apparent in tobacco leaves inoculated with DC3000(pBBR.Gus), regardless of the sampling period (Fig. 4c
). When the bacterial population was monitored during this experiment, the DC3000(pDCalgDP) and DC3000(pBBR.Gus) populations showed a decline during the 612 h sampling period (Fig. 4d
). This is possibly due to the accumulation of toxic defence compounds that are produced during the HR.
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Production of in tobacco inoculated with DC3000
When P. syringae was exposed to compounds that are known to generate ROS, the expression of algT was activated in vitro (Keith & Bender, 1999). The algT gene encodes
22, an alternative sigma factor that is required for expression of algD (Peñaloza-Vázquez et al., 1997
; Wozniak & Ohman, 1994
). Since alginate gene expression is known to be activated by ROS in vitro, we reasoned that these signals might also activate alginate production in the plant. For example, the algD : : uidA fusion was transcriptionally activated when tobacco was infiltrated with DC3000(pDCalgDP) (Fig. 4c
), but not with DC3000-hrcC(pDCalgDP) (Fig. 4e
). Since DC3000-hrcC does not induce an HR, these results suggest that the algD promoter is activated by signals generated prior to the HR, and these signals are absent or reduced when the HR is not elicited. To explore this hypothesis, pathogen-inoculated tobacco leaves were infiltrated with NBT, which facilitates the detection of
, one of the primary oxygen species produced during the HR (Baker & Orlandi, 1995
). When NBT reacts with
, a dark blue insoluble formazan compound is produced (Beyer & Fridovich, 1987
), which identifies the regions of superoxide formation in leaf tissue.
is thought to be the major oxidant species responsible for reducing NBT to formazan (Doke, 1983
; Fryer et al., 2002
). A transient production of
was observed in tobacco leaves inoculated with DC3000 and DC3000-hrcC at 4 h p.i. (Fig. 5
). However, in leaves inoculated with DC3000, a second burst of
production was detected at 810 h p.i. (Fig. 5
); this second phase of
accumulation was visibly reduced in tobacco leaves inoculated with DC3000-hrcC (Fig. 5
).
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DISCUSSION |
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In many plantpathogen interactions, protection from pathogen invasion is controlled by plant disease resistance (R) genes (Flor, 1971). R genes activate defence reactions by recognizing the presence of a corresponding avirulence (avr) gene of pathogen origin. The PtoR resistance gene in tomato encodes a serine-threonine protein kinase and confers resistance to isolates of P. syringae that express the avirulence gene avrPto, including strain DC3000 (Martin et al., 1993
; van Dijk et al., 1999
). When P. syringae pv. tomato DC3000 was spray-inoculated onto tomato Rio Grande-PtoR, an HR was visible when the tissue was examined by light microscopy (Fig. 3c, d
); however, necrotic cells were not visible without the aid of the microscope. This approach enabled us to monitor algD expression in internal and epiphytic populations of P. syringae pv. tomato DC3000. Interestingly, algD expression was observed in epiphytic populations of bacteria near plant cells undergoing an HR (Fig. 3e
) and in the apoplast near mesophyll cells that were responding hypersensitively (Fig. 3f
).
As expected, P. syringae pv. tomato DC3000 elicited a prominent HR when infiltrated into leaves of tobacco, which is not a host for this pathogen (Fig. 4a). Furthermore, algD was expressed in tobacco leaves undergoing the HR (Fig. 4c
), although algD expression was delayed and lower than that observed for the hrpA : : uidA fusion (Fig. 4c
). The lag period preceding algD expression in tobacco suggested that signals that are evolved during the onset of the HR might stimulate alginate gene expression. This was further investigated by analysing algD expression in DC3000-hrcC, which does not elicit an HR in tobacco leaves (Fig. 4b
). The hrpA : : uidA fusion in DC3000-hrcC was strongly induced in the absence of an HR (Fig. 4e
); this is consistent with regulation of hrpA transcription via the hrpL promoter, which should be functional in the hrcC mutant (Collmer et al., 2000
). However, we found that algD expression was negligible in tobacco leaves inoculated with DC3000-hrcC (Fig. 4e
), which suggests that manifestation of the HR is a prerequisite for algD transcription in tobacco leaves inoculated with P. syringae pv. tomato DC3000. These results also suggest that the hrpA and algD promoters respond to different signals in the DC3000tobacco interaction.
The HR is often associated with an oxidative burst, which involves the production of potentially cytotoxic quantities of H2O2 and (Wojtaszek, 1997
). This phenomenon was first identified in mammalian phagocytes and was described as the respiratory burst (Segal & Abo, 1993
). It is tempting to speculate that the ROS produced during the oxidative burst may function as signals for alginate gene induction in planta. The oxidative burst is generally described as a biphasic phenomenon in plant cells. During phase I, an immediate and transient production of ROS occurs and is non-specifically stimulated by compatible, incompatible and saprophytic bacteria (Baker & Orlandi, 1995
). We speculate that phase I production of ROS occurred at approximately 4 h p.i., since NBT staining (Fig. 5
) indicated the presence of
in tobacco leaves infiltrated with both DC3000 (produces HR) and DC3000-hrcC (no HR). Furthermore, the detection of ROS at 4 h p.i. is consistent with the timing of phase I in previous studies (Wojtaszek, 1997
). Phase II is characterized by a more prolonged production of ROS and is stimulated by incompatible bacteria that induce an HR (Baker & Orlandi, 1995
; Levine et al., 1994
). In our studies, the wild-type DC3000 is incompatible on tobacco, produces an HR and induces a second phase of ROS at 810 h p.i. (Fig. 5
). We hypothesize that this second burst (phase II) of
stimulates expression of the algD gene, either directly or indirectly. Since the DC3000-hrcC mutant does not elicit an HR in tobacco and does not induce phase II production of ROS, the signals for triggering algD expression may be absent or suboptimal in the DC3000-hrcC/tobacco interaction. In a highly relevant study, hrp mutants of P. syringae pv. tomato DC3000 failed to elicit significant levels of ROS in an incompatible interaction (Grant et al., 2000
), which is consistent with our results.
It is likely that ROS also function as signals for algD activation in tomato Rio Grande-PtoR inoculated with P. syringae pv. tomato DC3000. The interaction of AvrPto, which is present in DC3000, with the PtoR-encoded kinase initiates multiple defence pathways, including the production of ROS and elicitation of the HR (Jin & He, 2001; Sessa & Martin, 2000
). Chandra et al. (1996)
used tomato suspension cells to follow the oxidative burst in tomato lines challenged with P. syringae pv. tomato. Their results demonstrate that phase II of the oxidative burst is dependent on co-expression of PtoR in the tomato host and avrPto in the pathogen, an interaction that results in the HR (Chandra et al., 1996
). In Rio Grande-PtoS, the second oxidative burst is either absent or occurs at sublethal levels, which do not have an impact on the multiplication of the bacteria.
Although the plant signals that stimulate alginate production have not been identified, ROS are likely signals since they are known to stimulate alginate production in P. syringae and P. aeruginosa in vitro (Keith & Bender, 1999; Mathee et al., 1999
). Furthermore, Venisse et al. (2001)
recently demonstrated that a sustained production of ROS was required for initiation of necrotic lesion development by Erwinia amylovora in pear. Consequently, ROS may have a role in compatible interactions and could trigger alginate production in selected hostpathogen combinations. Further studies designed to identify the specific ROS that trigger algD expression are under way.
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ACKNOWLEDGEMENTS |
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Received 7 November 2002;
revised 7 February 2003;
accepted 13 February 2003.